Human phosphatase RET31, and variants thereof

ABSTRACT

The present invention provides novel polynucleotides encoding human phosphatase polypeptides, fragments and homologues thereof. Also provided are vectors, host cells, antibodies, and recombinant and synthetic methods for producing said polypeptides. The invention further relates to diagnostic and therapeutic methods for applying these novel human phosphatase polypeptides to the diagnosis, treatment, and/or prevention of various diseases and/or disorders related to these polypeptides, particularly cardiovascular diseases and/or disorders. The invention further relates to screening methods for identifying agonists and antagonists of the polynucleotides and polypeptides of the present invention.

This application is a divisional application of non-provisionalapplication U.S. Ser. No. 10/029,345, filed Dec. 20, 2001 now U.S. Pat.No. 7,153,678, which claims benefit to provisional application U.S. Ser.No. 60/256,868, filed Dec. 20, 2000; to provisional application U.S.Ser. No. 60/280,186, filed Mar. 30, 2001; to provisional applicationU.S. Ser. No. 60/287,735, filed May 01, 2001, to provisional applicationU.S. Ser. No. 60/295,848, filed Jun. 05, 2001, and to provisionalapplication U.S. Ser. No. 60/300,465, filed Jun. 25, 2001.

FIELD OF THE INVENTION

The present invention provides novel polynucleotides encoding humanphosphatase polypeptides, fragments and homologues thereof. Alsoprovided are vectors, host cells, antibodies, and recombinant andsynthetic methods for producing said polypeptides. The invention furtherrelates to diagnostic and therapeutic methods for applying these novelhuman phosphatase polypeptides to the diagnosis, treatment, and/orprevention of various diseases and/or disorders related to thesepolypeptides, particularly cardiovascular diseases and/or disorders. Theinvention further relates to screening methods for identifying agonistsand antagonists of the polynucleotides and polypeptides of the presentinvention.

BACKGROUND OF THE INVENTION

Phosphorylation of proteins is a fundamental mechanism for regulatingdiverse cellular processes. While the majority of proteinphosphorylation occurs at serine and threonine residues, phosphorylationat tyrosine residues is attracting a great deal of interest since thediscovery that many oncogene products and growth factor receptorspossess intrinsic protein tyrosine kinase activity. The importance ofprotein tyrosine phosphorylation in growth factor signal transduction,cell cycle progression and neoplastic transformation is now wellestablished (Hunter et al., Ann. Rev. Biochem. 54:987-930 (1985),Ullrich et al., Cell 61:203-212 (1990), Nurse, Nature 344:503-508(1990), Cantley et al, Cell 64:281-302 (1991)).

Biochemical studies have shown that phosphorylation on tyrosine residuesof a variety of cellular proteins is a dynamic process involvingcompeting phosphorylation and dephosphorylation reactions. Theregulation of protein tyrosine phosphorylation is mediated by thereciprocal actions of protein tyrosine kinases (PTKases) and proteintyrosine phosphatases (PTPases). The tyrosine phosphorylation reactionsare catalyzed by PTKases. Tyrosine phosphorylated proteins can bespecifically dephosphorylated through the action of PTPases. The levelof protein tyrosine phosphorylation of intracellular substances isdetermined by the balance of PTKase and PTPase activities. (Hunter, T.,Cell 58:1013-1016 (1989)).

The protein tyrosine kinases (PTKases) are a large family of proteinsthat includes many growth factor receptors and potential oncogenes.(Hanks et al., Science 241:42-52 (1988)). Many PTKases have been linkedto initial signals required for induction of the cell cycle (Weaver etal., Mol. Cell. Biol. 11, 9:4415-4422 (1991)). PTKases comprise adiscrete family of enzymes having common ancestry with, but majordifferences from, serine/threonine-specific protein kinases (Hanks etal., supra). The mechanisms leading to changes in activity of PTKasesare best understood in the case of receptor-type PTKases having atransmembrane topology (Ullrich et al. (1990) supra). The binding ofspecific ligands to the extracellular domain of members of receptor-typePTKases is thought to induce their oligomerization leading to anincrease in tyrosine kinase activity and activation of the signaltransduction pathways (Ullrich et al., (1990) supra). Deregulation ofkinase activity through mutation or overexpression is a well establishedmechanism for cell transformation (Hunter et al., (1985) supra; Ullrichet al., (1990) supra).

The protein phosphatases are composed of at least two separate anddistinct families (Hunter, T. (1989) supra) the protein serine/threoninephosphatases and the protein tyrosine phosphatases (PTPases).

The protein tyrosine phosphatases (PTPases) are a family of proteinsthat have been classified into two subgroups. The first subgroup is madeup of the low molecular weight, intracellular enzymes that contain asingle conserved catalytic phosphatase domain. All known intracellulartype PTPases contain a single conserved catalytic phosphatase domain.Examples of the first group of PTPases include (1) placental PTPase 1B(Charbonneau et al., Proc. Natl. Acad. Sci. USA 86:5252-5256 (1989);Chernoff et al., Proc. Natl. Acad. Sci. USA 87:2735-2789 (1989)), (2)T-cell PTPase (Cool et al., Proc. Natl. Acad. Sci. USA 86:5257-5261(1989)), (3) rat brain PTPase (Guan et al., Proc. Natl. Acad. Sci. USA87:1501-1502 (1990)), (4) neuronal phosphatase (STEP) (Lombroso et al.,Proc. Natl. Acad. Sci. USA 88:7242-7246 (1991)), and (5) cytoplasmicphosphatases that contain a region of homology to cytoskeletal proteins(Gu et al., Proc. Natl. Acad. Sci. USA 88:5867-57871 (1991); Yang etal., Proc. Natl. Acad. Sci. USA 88:5949-5953 (1991)).

Enzymes of this class are characterized by an active site motif of CX₅R.Within ths motif the Cysteine sulfur acts as a nucleophile which cleavesthe P—O bond and releases the phosphate; the Arginine interacts with thephosphate and facilitates nucleophic attack. In many cases the Cysteineis preceded by a Histidine and the Arginine is followed by a Serine orThreonine. In addition, an Aspartate residue located 20 or more aminoacids N terminal to the Cysteine acts as a general acid during cleavage[Fauman, 1996].

The second subgroup of protein tyrosine phosphatases is made up of thehigh molecular weight, receptor-linked PTPases, termed R-PTPases.R-PTPases consist of a) an intracellular catalytic region, b) a singletransmembrane segment, and c) a putative ligand-binding extracellulardomain (Gebbink et al., supra).

The structures and sizes of the c) putative ligand-binding extracellular“receptor” domains of R-PTPases are quite divergent. In contrast, the a)intracellular catalytic regions of R-PTPases are highly homologous. AllRPTPases have two tandemly duplicated catalytic phosphatase homologydomains, with the prominent exception of an R-PTPase termed HPTP.beta.,which has “only one catalytic phosphatase domain. (Tsai et al., J. Biol.Chem. 266(16):10534-10543 (1991)).

One example of R-PTPases are the leukocyte common antigens (LCA) (Ralph,S. J., EMBO J. 6:1251-1257 (1987)). LCA is a family of high molecularweight glycoproteins expressed on the surface of all leukocytes andtheir hemopoietic progenitors (Thomas, Ann. Rev. Immunol. 7:339-369(1989)). A remarkable degree of similarity is detected with the sequenceof LCA from several species (Charbonneau et al., Proc. Natl. Acad. Sci.USA 85:7182-7186 (1988)). LCA is referred to in the literature bydifferent names, including T200 (Trowbridge et al., Eur. J. Immunol.6:557-562 (1962)), B220 for the B cell form (Coffman et al., Nature289:681-683 (1981)), the mouse allotypic marker Ly-5 (Komuro et al.,Immunogenetics 1:452-456 (1975)), and more recently CD45 (Cobbold etal., Leucocyte Typing III, ed. A. J. McMichael et al., pp. 788-803(1987)).

Several studies suggest that CD45 plays a critical role in T cellactivation. These studies are reviewed in Weiss A., Ann. Rev. Genet.25:487-510 (1991). In one study, T-cell clones that were mutagenized byNSG and selected for their failure to express CD45 had impairedresponses to T-cell receptor stimuli (Weaver et al., (1991) supra).These T-cell clones were functionally defective in their responses tosignals transmitted through the T cell antigen receptor, includingcytolysis of appropriate targets, proliferation, and lymphokineproduction (Weaver et al., (1991) supra).

Other studies indicate that the PTPase activity of CD45 plays a role inthe activation of pp56.sup.lck, a lymphocyte-specific PTKase (Mustelinet al., Proc. Natl. Acad. Sci. USA 86:6302-6306 (1989); Ostergaard etal., Proc. Natl. Acad. Sci. USA 86:8959-8963 (1989)). These authorshypothesized that the phosphatase activity of CD45 activatespp56.sup.lck by dephosphorylation of a C-terminal tyrosine residue,which may, in turn, be related to T-cell activation.

Another example of R-PTPases is the leukocyte common antigen relatedmolecule (LAR) (Streuli et al., J. Exp. Med. 168:1523-1530 (1988)). LARwas initially identified as a homologue of LCA (Streuli et al., supra).Although the a) intracellular catalytic region of the LAR moleculecontains two catalytic phosphatase homology domains (domain I and domainII), mutational analyses suggest that only domain I has catalyticphosphatase activity, whereas domain II is enzymatically inactive(Streuli et al., EMBO J. 9(8):2399-2407 (1990)). Chemically induced LARmutants having tyrosine at amino acid position 1379 changed to aphenylalanine are temperature-sensitive (Tsai et al., J. Biol. Chem.266(16):10534-10543 (1991)).

A new mouse R-PTP, designated mRPTP.mu., has been cloned which has a) anextracellular domain that shares some structural motifs with LAR.(Gebbink et al., (1991) supra). In addition, these authors have clonedthe human homologue of RPTP.mu. and localized the gene on humanchromosome 18.

Two Drosophila PTPases, termed DLAR and DPTP, have been predicted basedon the sequences of cDNA clones (Streuli et al., Proc. Natl. Acad. Sci.USA 86:8698-8702 (1989)). cDNAs coding for another Drosophila R-PTPase,termed DPTP 99A, have been cloned and characterized (Hariharan et al.,Proc. Natl. Acad. Sci. USA 88:11266-11270 (1991)).

Other examples of R-PTPases include R-PTPase-.alpha., .beta., gamma.,and .zeta. (Krueger et al., EMBO J. 9:3241-3252 (1990), Sap et al.,Proc. Natl. Acad. Sci. USA 87:6112-6116 (1990), Kaplan et al., Proc.Natl. Acad. Sci. USA 87:7000-7004 (1990), Jirik et al., FEBS Lett.273:239-242 (1990); Mathews et al., Proc. Natl. Acad. Sci. USA87:4444-4448 (1990), Ohagi et al., Nucl. Acids Res. 18:7159 (1990)).Published application WO92/01050 discloses human R-PTPase-.alpha.,.beta. and .gamma., and reports on the nature of the structuralhomologies found among the conserved domains of these three R-PTPasesand other members of this protein family. The murine R-PTPase-.alpha.has 794 amino acids, whereas the human R-PTPase-.alpha. has 802 aminoacids. R-PTPase-.alpha. has an intracellular domain homologous to thecatalytic domains of other tyrosine phosphatases. The 142 amino acidextracellular domain (including signal peptide of RPTPase-.alpha.) has ahigh serine and threonine content (32%) and 8 potential N-glycosylationsites. cDNA clones have been produced that code for theR-PTPase-.alpha., and R-PTPase-.alpha. has been expressed fromeukaryotic hosts. Northern analysis has been used to identify thenatural expression of R-PTPase-.alpha. in various cells and tissues. Apolyclonal antibody to R-PTPase-.alpha. has been produced byimmunization with a synthetic peptide of R-PTPase-.alpha., whichidentifies a 130 kDa protein in cells transfected with a cDNA cloneencoding a portion of R-PTPase-.alpha.

Another example of R-PTPases is HePTP. (Jirik et al, FASEB J. 4:82082(1990) Abstract 2253). Jirik et al. screened a cDNA library derived froma hepatoblastoma cell line, HepG2, with a probe encoding the two PTPasedomains of LCA, and discovered a cDNA clone encoding a new RPTPase,named HePTP. The HePTP gene appeared to be expressed in a variety ofhuman and murine cell lines and tissues.

Since the initial purification, sequencing, and cloning of a PTPase,additional potential PTPases have been identified at a rapid pace. Thenumber of different PTPases that have been identified is increasingsteadily, leading to speculations that this family may be as large asthe PTKase family (Hunter (1989) supra).

Conserved amino acid sequences in the catalytic domains of known PTPaseshave been identified and defined (Krueger et al., EMBO J. 9:3241-3252(1990) and Yi et al., Mol. Cell. Biol. 12:836-846 (1992), which areincorporated herein by reference.) These amino acid sequences aredesignated “consensus sequences” herein.

Yi et al. aligned the catalytic phosphatase domain sequences of thefollowing PTPases: LCA, PTP1B, TCPTP, LAR, DLAR, and HPTP.alpha.,HPTP.beta., and HPTP.gamma. This alignment includes the following“consensus sequences” (Yi et al., supra, FIG. 2(A), lines 1 and 2):DYINAS/N (SEQ ID NO:77), CXXYWP (SEQ ID NO:78), and I/VVMXXXXE (SEQ IDNO:79).

Krueger et al., aligned the catalytic phosphatase domain sequences ofPTP1B, TCPTP, LAR, LCA, HPTP.alpha., .beta., .gamma., .GAMMA., .delta.,.epsilon. and .zeta. and DLAR and DPTP. This alignment includes thefollowing “consensus sequences: (Krueger et al., supra, FIG. 7, lines 1and 2): D/NYINAS/N (SEQ ID NO:80), CXXYWP (SEQ ID NO:81), and I/VVMXXXXE(SEQ ID NO:82).

It is becoming clear that dephosphorylation of tyrosine residues can byitself function as an important regulatory mechanism. Dephosphorylationof a C-terminal tyrosine residue has been shown to activate tyrosinekinase activity in the case of the src family of tyrosine kinases(Hunter, T. Cell 49:1-4 (1987)). Tyrosine dephosphorylation has beensuggested to be an obligatory step in the mitotic activation of thematuration-promoting factor (MPF) kinase (Morla et al., Cell 58:193-203(1989)). These observations point out the need in the art forunderstanding the mechanisms that regulate tyrosine phosphataseactivity.

Modulators (inhibitors or activators) of human phosphatase expression oractivity could be used to treat a subject with a disorder characterizedby aberrant phosphatase expression or activity or by decreasedphosphorylation of a phosphatase substrate protein. Examples of suchdisorders include but are not limited to: an immune, anti-proliferative,proliferative (e.g. cancer), metabolic (e.g. diabetes or obesity), bone(e.g., osteoporosis), neural, and/or cardiovascular diseases and/ordisorders, in addition to, viral pathogenesis.

It is clear that further analysis of structure-function relationshipsamong PTPases are needed to gain important understanding of themechanisms of signal transduction, cell cycle progression and cellgrowth, and neoplastic transformation.

The present invention also relates to recombinant vectors, which includethe isolated nucleic acid molecules of the present invention, and tohost cells containing the recombinant vectors, as well as to methods ofmaking such vectors and host cells, in addition to their use in theproduction of human phosphatase polypeptides or peptides usingrecombinant techniques. Synthetic methods for producing the polypeptidesand polynucleotides of the present invention are provided. Also providedare diagnostic methods for detecting diseases, disorders, and/orconditions related to the human phosphatase polypeptides andpolynucleotides, and therapeutic methods for treating such diseases,disorders, and/or conditions. The invention further relates to screeningmethods for identifying binding partners of the polypeptides.

BRIEF SUMMARY OF THE INVENTION

The present invention provides isolated nucleic acid molecules, thatcomprise, or alternatively consist of, a polynucleotide encoding thehuman BMY_HPP 1 phosphatase protein having the amino acid sequence shownas SEQ ID NO:150, or the amino acid sequence encoded by the cDNA cloneBMY_HPP 1, deposited as ATCC Deposit Number PTA-3949 on Dec. 22, 2001.

The present invention provides isolated nucleic acid molecules, thatcompnse, or alternatively consist of, a polynucleotide encoding theBMY_HPP2 phosphatase protein having the amino acid sequence shown as SEQID NO:152, or the amino acid sequence encoded by the cDNA cloneBMY_HPP2, deposited as ATCC Deposit Number PTA-3949 on Dec. 22, 2001.

The present invention provides isolated nucleic acid molecules, thatcomprise, or alternatively consist of, a polynucleotide encoding thehuman BMY_HPP5 phosphatase protein having the amino acid sequence shownas SEQ ID NO:42, or the amino acid sequence encoded by the cDNA clone,BMY_HPP5 (also referred to as 71C-5-E2), deposited as ATCC DepositNumber PTA-2966 on Jan. 24, 2001.

The present invention provides isolated nucleic acid molecules, thatcomprise, or alternatively consist of, a polynucleotide encoding thehuman RET31 phosphatase protein having the amino acid sequence shown asSEQ ID NO:109, or the amino acid sequence encoded by the cDNA clone,RET31 (also referred to as 1hrTNF031, and/or Clone 31), deposited asATCC Deposit Number PTA-3434 on Jun. 7, 2001.

The present invention provides isolated nucleic acid molecules, thatcomprise, or alternatively consist of, a polynucleotide encoding themouse RET31 phosphatase protein having the amino acid sequence shown asSEQ ID NO:114.

The present invention also relates to recombinant vectors, which includethe isolated nucleic acid molecules of the present invention, and tohost cells containing the recombinant vectors, as well as to methods ofmaking such vectors and host cells, in addition to their use in theproduction of human phosphatase polypeptides or peptides usingrecombinant techniques. Synthetic methods for producing the polypeptidesand polynucleotides of the present invention are provided. Also providedare diagnostic methods for detecting diseases, disorders, and/orconditions related to the human phosphatase polypeptides andpolynucleotides, and therapeutic methods for treating such diseases,disorders, and/or conditions. The invention further relates to screeningmethods for identifying binding partners of the polypeptides.

The invention further provides an isolated BMY_HPP1 human phosphatasepolypeptide having an amino acid sequence encoded by a polynucleotidedescribed herein.

The invention further provides an isolated BMY_HPP2 human phosphatasepolypeptide having an amino acid sequence encoded by a polynucleotidedescribed herein.

The invention further provides an isolated BMY_HPP5 human phosphatasepolypeptide having an amino acid sequence encoded by a polynucleotidedescribed herein.

The invention further provides an isolated RET31 human phosphatasepolypeptide having an amino acid sequence encoded by a polynucleotidedescribed herein.

The invention further provides an isolated RET31 mouse phosphatasepolypeptide having an amino acid sequence encoded by a polynucleotidedescribed herein.

The invention further relates to a polynucleotide encoding a polypeptidefragment of SEQ ID NO:150, 152, 8, 10, 42, or 109, or a polypeptidefragment encoded by the cDNA sequence included in the deposited clone,which is hybridizable to SEQ ID NO:149, 151, 7, 9, 41, or 108.

The invention further relates to a polynucleotide encoding a polypeptidedomain of SEQ ID NO:150, 152, 8, 10, 42, or 109 or a polypeptide domainencoded by the cDNA sequence included in the deposited clone, which ishybridizable to SEQ ID NO:149, 151, 7, 9, 41, or 108.

The invention further relates to a polynucleotide encoding a polypeptideepitope of SEQ ID NO:150, 152, 8, 10, 42, or 109 or a polypeptideepitope encoded by the cDNA sequence included in the deposited clone,which is hybridizable to SEQ ID NO:149, 151, 7, 9, 41, or 108.

The invention further relates to a polynucleotide encoding a polypeptideof SEQ ID NO:150, 152, 8, 10, 42, or 109 or the cDNA sequence includedin the deposited clone, which is hybridizable to SEQ ID NO:149, 151, 7,9, 41, or 108, having biological activity.

The invention further relates to a polynucleotide which is a variant ofSEQ ID NO:149, 151, 7, 9, 41, or 108.

The invention further relates to a polynucleotide which is an allelicvariant of SEQ ID NO:149, 151, 7, 9, 41, or 108.

The invention further relates to a polynucleotide which encodes aspecies homologue of the SEQ ID NO:150, 152, 8, 10, 42, or 109.

The invention further relates to a polynucleotide which represents thecomplimentary sequence (antisense) of SEQ ID NO:149, 151, 7, 9, 41, or108.

The invention further relates to a polynucleotide capable of hybridizingunder stringent conditions to any one of the polynucleotides specifiedherein, wherein said polynucleotide does not hybridize under stringentconditions to a nucleic acid molecule having a nucleotide sequence ofonly A residues or of only T residues.

The invention further relates to an isolated nucleic acid molecule ofSEQ ID NO:150, 152, 8, 10, 42, or 109, wherein the polynucleotidefragment comprises a nucleotide sequence encoding a human phosphataseprotein.

The invention further relates to an isolated nucleic acid molecule ofSEQ ID NO: 149, 151, 7, 9, 41, or 108 wherein the polynucleotidefragment comprises a nucleotide sequence encoding the sequenceidentified as SEQ ID NO:150, 152, 8, 10, 42, or 109 or the polypeptideencoded by the cDNA sequence included in the deposited clone, which ishybridizable to SEQ ID NO:149, 151, 7, 9, 41, or 108.

The invention further relates to an isolated nucleic acid molecule of ofSEQ ID NO: 149, 151, 7, 9, 41, or 108, wherein the polynucleotidefragment comprises the entire nucleotide sequence of SEQ ID NO:149, 151,7, 9, 41, or 108 or the cDNA sequence included in the deposited clone,which is hybridizable to SEQ ID NO:149, 151, 7, 9, 41, or 108.

The invention further relates to an isolated nucleic acid molecule ofSEQ ID NO:1, wherein the nucleotide sequence comprises sequentialnucleotide deletions from either the C-terminus or the N-terminus.

The invention further relates to an isolated polypeptide comprising anamino acid sequence that comprises a polypeptide fragment of SEQ IDNO:150, 152, 8, 10, 42, or 109 or the encoded sequence included in thedeposited clone.

The invention further relates to a polypeptide fragment of SEQ IDNO:150, 152, 8, 10, 42, or 109 or the encoded sequence included in thedeposited clone, having biological activity.

The invention further relates to a polypeptide domain of SEQ ID NO:150,152, 8, 10, 42, or 109 or the encoded sequence included in the depositedclone.

The invention further relates to a polypeptide epitope of SEQ ID NO:150,152, 8, 10, 42, or 109 or the encoded sequence included in the depositedclone.

The invention further relates to a full length protein of SEQ ID NO:150,152, 8, 10, 42, or 109 or the encoded sequence included in the depositedclone.

The invention further relates to a variant of SEQ ID NO:150, 152, 8, 10,42, or 109.

The invention further relates to an allelic variant of SEQ ID NO:150,152, 8, 10, 42, or 109. The invention further relates to a specieshomologue of SEQ ID NO:150, 152, 8, 10, 42, or 109.

The invention further relates to the isolated polypeptide of of SEQ IDNO:150, 152, 8, 10, 42, or 109, wherein the full length proteincomprises sequential amino acid deletions from either the C-terminus orthe N-terminus.

The invention further relates to an isolated antibody that bindsspecifically to the isolated polypeptide of SEQ ID NO:150, 152, 8, 10,42, or 109.

The invention further relates to a method for preventing, treating, orameliorating a medical condition, comprising administering to amammalian subject a therapeutically effective amount of the polypeptideof SEQ ID NO:150, 152, 8, 10, 42, or 109 or the polynucleotide of SEQ IDNO:149, 151, 7, 9, 41, or 108.

The invention further relates to a method of diagnosing a pathologicalcondition or a susceptibility to a pathological condition in a subjectcomprising the steps of (a) determining the presence or absence of amutation in the polynucleotide of SEQ ID NO:149, 151, 7, 9, 41, or 108;and (b) diagnosing a pathological condition or a susceptibility to apathological condition based on the presence or absence of saidmutation.

The invention further relates to a method of diagnosing a pathologicalcondition or a susceptibility to a pathological condition in a subjectcomprising the steps of (a) determining the presence or amount ofexpression of the polypeptide of of SEQ ID NO:150, 152, 8, 10, 42, or109 in a biological sample; and diagnosing a pathological condition or asusceptibility to a pathological condition based on the presence oramount of expression of the polypeptide.

The invention further relates to a method for identifying a bindingpartner to the polypeptide of SEQ ID NO:150, 152, 8, 10, 42, or 109comprising the steps of (a) contacting the polypeptide of SEQ ID NO:150,152, 8, 10, 42, or 109 with a binding partner; and (b) determiningwhether the binding partner effects an activity of the polypeptide.

The invention further relates to a gene corresponding to the cDNAsequence of SEQ ID NO:149, 151, 7, 9, 41, or 108.

The invention further relates to a method of identifying an activity ina biological assay, wherein the method comprises the steps of expressingSEQ ID NO:149, 151, 7, 9, 41, or 108 in a cell, (b) isolating thesupernatant; (c) detecting an activity in a biological assay; and (d)identifying the protein in the supernatant having the activity.

The invention further relates to a process for making polynucleotidesequences encoding gene products having altered activity selected fromthe group consisting of SEQ ID NO:150, 152, 8, 10, 42, or 109 activitycomprising the steps of (a) shuffling a nucleotide sequence of SEQ IDNO:149, 151, 7, 9, 41, or 108, (b) expressing the resulting shufflednucleotide sequences and, (c) selecting for altered activity selectedfrom the group consisting of SEQ ID NO:150, 152, 8, 10, 42, or 109activity as compared to the activity selected from the group consistingof SEQ ID NO:150, 152, 8, 10, 42, or 109 activity of the gene product ofsaid unmodified nucleotide sequence.

The invention further relates to a shuffled polynucleotide sequenceproduced by a shuffling process, wherein said shuffled DNA moleculeencodes a gene product having enhanced tolerance to an inhibitor of anyone of the activities selected from the group consisting of SEQ IDNO:150, 152, 8, 10, 42, or 109 activity.

The invention further relates to a method for preventing, treating, orameliorating a medical condition with the polypeptide provided as SEQ IDNO:150, 152, 8, 10, 42, or 109, in addition to, its encoding nucleicacid, wherein the medical condition is a condition related to aberrantphosphatase activity.

The invention further relates to a method of identifying a compound thatmodulates the biological activity of a phosphatase, comprising the stepsof, (a) combining a candidate modulator compound with a phosphatasehaving the sequence set forth in one or more of SEQ ID NO:150, 152, 8,10, 42, or 109; and measuring an effect of the candidate modulatorcompound on the activity of a phosphatase.

The invention further relates to a method of identifying a compound thatmodulates the biological activity of a phosphatase, comprising the stepsof, (a) combining a candidate modulator compound with a host cellexpressing a phosphatase having the sequence as set forth in SEQ IDNO:150, 152, 8, 10, 42, or 109; and, (b) measuring an effect of thecandidate modulator compound on the activity of the expressed aphosphatase.

The invention further relates to a method of identifying a compound thatmodulates the biological activity of a phosphatase, comprising the stepsof, (a) combining a candidate modulator compound with a host cellcontaining a vector described herein, wherein a phosphatase is expressedby the cell; and, (b) measuring an effect of the candidate modulatorcompound on the activity of the expressed a phosphatase.

The invention further relates to a method of screening for a compoundthat is capable of modulating the biological activity of a phosphatase,comprising the steps of: (a) providing a host cell described herein; (b)determining the biological activity of a phosphatase in the absence of amodulator compound; (c) contacting the cell with the modulator compound;and (d) determining the biological activity of a phosphatase in thepresence of the modulator compound; wherein a difference between theactivity of a phosphatase in the presence of the modulator compound andin the absence of the modulator compound indicates a modulating effectof the compound.

The invention further relates to a compound that modulates thebiological activity of human a phosphatase as identified by the methodsdescribed herein.

The invention also relates to in silico screening methods including insilico docking and methods of structure based drug design which utilizethe three dimensional coordinates of BMY_HPP1 (FIG. 28, Table VIII).Also provided are methods of identifying modulators of BMY_HPP1 thatinclude modulator building or searching utilizing computer programs andalgorithms. In an embodiment of the invention a method is provided fordesigning potential modulators of BMY_HPP1 comprising any combination ofsteps which utilize said three dimensional structure to design or selectpotential modulators.

The present invention also provides structure coordinates of thehomology model of BMY_HPP1. The complete coordinates are listed in TableVIII and visualized in FIG. 28. The model present in this inventionfurther provides a basis for designing stimulators and inhibitors orantagonists of one or more of the biological functions of BMY_HPP1, orof mutants with altered specificity.

The invention also relates to in silico screening methods including insilico docking and methods of structure based drug design which utilizethe three dimensional coordinates of BMY_HPP2 (FIG. 32, Table IX). Alsoprovided are methods of identifying modulators of BMY_HPP2 that includemodulator building or searching utilizing computer programs andalgorithms. In an embodiment of the invention a method is provided fordesigning potential modulators of BMY_HPP2 comprising any combination ofsteps which utilize said three dimensional structure to design or selectpotential modulators.

The present invention also provides structure coordinates of thehomology model of BMY_HPP2. The complete coordinates are listed in TableIX and visualized in FIG. 32. The model present in this inventionfurther provides a basis for designing stimulators and inhibitors orantagonists of one or more of the biological functions of BMY_HPP2, orof mutants with altered specificity.

The invention also relates to in silico screening methods including insilico docking and methods of structure based drug design which utilizethe three dimensional coordinates of BMY_HPP5 (FIG. 38, Table X). Alsoprovided are methods of identifying modulators of BMY_HPP5 that includemodulator building or searching utilizing computer programs andalgorithms. In an embodiment of the invention a method is provided fordesigning potential modulators of BMY_HPP5 comprising any combination ofsteps which utilize said three dimensional structure to design or selectpotential modulators.

The present invention also provides structure coordinates of thehomology model of BMY_HPP5. The complete coordinates are listed in TableX and visualized in FIG. 38. The model present in this invention furtherprovides a basis for designing stimulators and inhibitors or antagonistsof one or more of the biological functions of BMY_HPP5, or of mutantswith altered specificity.

The invention also provides a machine readable storage medium whichcomprises the structure coordinates of BMY_HPP1, including all or anyparts conserved active site regions. Such storage medium encoded withthese data are capable of displaying on a computer screen or similarviewing device, a three-dimensional graphical representation of amolecule or molecular complex which comprises said regions or similarlyshaped homologous regions.

The invention also provides methods for designing, evaluating andidentifying compounds which bind to all or parts of the aforementionedregions. The methods include three dimensional model building (homologymodeling) and methods of computer assisted-drug design which can be usedto identify compounds which bind or modulate the forementioned regionsof the BMY_HPP1 polypeptide. Such compounds are potential inhibitors ofBMY_HPP1 or its homologues.

The invention also provides a machine readable storage medium whichcomprises the structure coordinates of BMY_HPP2, including all or anyparts conserved active site regions. Such storage medium encoded withthese data are capable of displaying on a computer screen or similarviewing device, a three-dimensional graphical representation of amolecule or molecular complex which comprises said regions or similarlyshaped homologous regions.

The invention also provides methods for designing, evaluating andidentifying compounds which bind to all or parts of the aforementionedregions. The methods include three dimensional model building (homologymodeling) and methods of computer assisted-drug design which can be usedto identify compounds which bind or modulate the forementioned regionsof the BMY_HPP2 polypeptide. Such compounds are potential inhibitors ofBMY_HPP2 or its homologues.

The invention also provides a machine readable storage medium whichcomprises the structure coordinates of BMY_HPP5, including all or anyparts conserved active site regions. Such storage medium encoded withthese data are capable of displaying on a computer screen or similarviewing device, a three-dimensional graphical representation of amolecule or molecular complex which comprises said regions or similarlyshaped homologous regions.

The invention also provides methods for designing, evaluating andidentifying compounds which bind to all or parts of the aforementionedregions. The methods include three dimensional model building (homologymodeling) and methods of computer assisted-drug design which can be usedto identify compounds which bind or modulate the forementioned regionsof the BMY_HPP5 polypeptide. Such compounds are potential inhibitors ofBMY_HPP5 or its homologues.

The invention also provides a computer for producing a three-dimensionalrepresentation of a molecule or molecular complex, wherein said moleculeor molecular complex comprises the structural coordinates of the modelBMY_HPP1 in accordance with Table VIII, or a three-dimensionalrepresentation of a homologue of said molecule or molecular complex,wherein said homologue comprises backbone atoms that have a root meansquare deviation from the backbone atoms of not more than 3.5 angstroms.wherein said computer comprises:

The invention also provides a machine-readable data storage medium,comprising a data storage material encoded with machine readable data,wherein the data is defined by the set of structure coordinates of themodel BMY_HPP1 according to Table VIII, or a homologue of said model,wherein said homologue comprises backbone atoms that have a root meansquare deviation from the backbone atoms of not more than 3.5 Å; aworking memory for storing instructions for processing saidmachine-readable data; a central-processing unit coupled to said workingmemory and to said machine-readable data storage medium for processingsaid machine readable data into said three-dimensional representation;and a display coupled to said central-processing unit for displayingsaid three-dimensional representation. The invention also provides saidcomputer wherein the machine-readable data storage medium is defined bythe set of structure coordinates of the model for BMY_HPP1 according toTable VIII, or a homologue of said molecule, said homologue having aroot mean square deviation from the backbone atoms of not more than 3.0Å.

The invention also provides a model comprising all or any part of themodel defined by structure coordinates of BMY_HPP1 according to TableVIII, or a mutant or homologue of said molecule or molecular complex.

The invention also provides a method for identifying a mutant ofBMY_HPP1 with altered biological properties, function, or reactivity,the method comprising the step selected from the group consisting of:Using the BMY_HPP1 model or a homologue of said model according to TableVIII, for the design of protein mutants with altered biological functionor properties.

The invention also provides a method for identifying structural andchemical features of BMY_HPP1 using the structural coordinates set forthin Table VIII, comprising any steps or combination of steps consistingof: employing identified structural or chemical features to design orselect compounds as potential BMY_HPP1 modulators; employing thethree-dimensional structural model to design or select compounds aspotential BMY_HPP1 modulators; synthesizing the potential BMY_HPP1modulators; and screening the potential BMY_HPP1 modulators in an assaycharacterized by binding of a protein to the BMY_HPP1. The inventionfurther provides said method wherein the potential BMY_HPP1 modulator isselected from a database. The invention further provides said methodwherein the potential BMY_HPP1 modulator is designed de novo. Theinvention further provides said method wherein the potential BMY_HPP1modulator is designed from a known modulator of activity.

The invention also provides a method for identifying a compound thatmodulates BMY_HPP1 activity, the method comprising any combination ofsteps of: Modeling test compounds that fit spatially into or near theactive site region defined by residues D161-Y162 and H189-C190-G193-R196of BMY_HPP1 as defined by structure coordinates according to Table VIII,or modeling test compounds that fit spatially into a three-dimensionalstructural model of the catalytic domain of BMY_HPP1, mutant homologueor portion thereof; using said structure coordinates or said active siteregion as set forth in prior claims to identify structural and chemicalfeatures; employing identified structural or chemical features to designor select compounds as potential BMY_HPP1 modulators includingsubstrates, antagonists and agonists; employing the three-dimensionalstructural model or the catalytic domain of BMY_HPP1 to design or selectcompounds as potential BMY_HPP1 inhibitors; screening the potentialBMY_HPP1 inhibitors in an assay characterized by binding of a testcompound to BMY_HPP1; and/or modifying or replacing one or more aminoacids from BMY_HPP1 including but not limited to the residuescorresponding to the active site region as set forth in prior claims ofBMY_HPP1 according to Table VIII.

The invention also provides a computer for producing a three-dimensionalrepresentation of a molecule or molecular complex, wherein said moleculeor molecular complex comprises the structural coordinates of the modelBMY_HPP2 in accordance with Table IX, or a three-dimensionalrepresentation of a homologue of said molecule or molecular complex,wherein said homologue comprises backbone atoms that have a root meansquare deviation from the backbone atoms of not more than 3.5 angstroms.wherein said computer comprises:

The invention also provides a machine-readable data storage medium,comprising a data storage material encoded with machine readable data,wherein the data is defined by the set of structure coordinates of themodel BMY_HPP2 according to Table IX, or a homologue of said model,wherein said homologue comprises backbone atoms that have a root meansquare deviation from the backbone atoms of not more than 3.5 Å; aworking memory for storing instructions for processing saidmachine-readable data; a central-processing unit coupled to said workingmemory and to said machine-readable data storage medium for processingsaid machine readable data into said three-dimensional representation;and a display coupled to said central-processing unit for displayingsaid three-dimensional representation. The invention also provides saidcomputer wherein the machine-readable data storage medium is defined bythe set of structure coordinates of the model for BMY_HPP2 according toTable IX, or a homologue of said molecule, said homologue having a rootmean square deviation from the backbone atoms of not more than 3.0 Å.

The invention also provides a model comprising all or any part of themodel defined by structure coordinates of BMY_HPP2 according to TableIX, or a mutant or homologue of said molecule or molecular complex.

The invention also provides a method for identifying a mutant ofBMY_HPP2 with altered biological properties, function, or reactivity,the method comprising the step selected from the group consisting of:Using the BMY_HPP2 model or a homologue of said model according to TableIX, for the design of protein mutants with altered biological functionor properties.

The invention also provides a method for identifying structural andchemical features of BMY_HPP2 using the structural coordinates set forthin Table IX, comprising any steps or combination of steps consisting of:employing identified structural or chemical features to design or selectcompounds as potential BMY_HPP2 modulators; employing thethree-dimensional structural model to design or select compounds aspotential BMY_HPP2 modulators; synthesizing the potential BMY_HPP2modulators; and screening the potential BMY_HPP2 modulators in an assaycharacterized by binding of a protein to the BMY_HPP2. The inventionfurther provides said method wherein the potential BMY_HPP2 modulator isselected from a database. The invention further provides said methodwherein the potential BMY_HPP2 modulator is designed de novo. Theinvention further provides said method wherein the potential BMY_HPP2modulator is designed from a known modulator of activity.

The invention also provides a method for identifying a compound thatmodulates BMY_HPP2 activity, the method comprising any combination ofsteps of: Modeling test compounds that fit spatially into or near theactive site region defined by residues residues D65, H94-C95, G98, andR101 of BMY_HPP2 as defined by structure coordinates according to TableIX, or modeling test compounds that fit spatially into athree-dimensional structural model of the catalytic domain of BMY_HPP2,mutant homologue or portion thereof; using said structure coordinates orsaid active site region as set forth in prior claims to identifystructural and chemical features; employing identified structural orchemical features to design or select compounds as potential BMY_HPP2modulators including substrates, antagonists and agonists; employing thethree-dimensional structural model or the catalytic domain of BMY_HPP2to design or select compounds as potential BMY_HPP2 inhibitors;screening the potential BMY_HPP2 inhibitors in an assay characterized bybinding of a test compound to BMY_HPP2; and/or modifying or replacingone or more amino acids from BMY_HPP2 including but not limited to theresidues corresponding to the active site region as set forth in priorclaims of BMY_HPP2 according to Table IX.

The invention also provides a computer for producing a three-dimensionalrepresentation of a molecule or molecular complex, wherein said moleculeor molecular complex comprises the structural coordinates of the modelBMY_HPP5 in accordance with Table X, or a three-dimensionalrepresentation of a homologue of said molecule or molecular complex,wherein said homologue comprises backbone atoms that have a root meansquare deviation from the backbone atoms of not more than 3.5 angstroms.wherein said computer comprises:

The invention also provides a machine-readable data storage medium,comprising a data storage material encoded with machine readable data,wherein the data is defined by the set of structure coordinates of themodel BMY_HPP5 according to Table X, or a homologue of said model,wherein said homologue comprises backbone atoms that have a root meansquare deviation from the backbone atoms of not more than 3.5 Å; aworking memory for storing instructions for processing saidmachine-readable data; a central-processing unit coupled to said workingmemory and to said machine-readable data storage medium for processingsaid machine readable data into said three-dimensional representation;and a display coupled to said central-processing unit for displayingsaid three-dimensional representation. The invention also provides saidcomputer wherein the machine-readable data storage medium is defined bythe set of structure coordinates of the model for BMY_HPP5 according toTable X, or a homologue of said molecule, said homologue having a rootmean square deviation from the backbone atoms of not more than 3.0 Å.

The invention also provides a model comprising all or any part of themodel defined by structure coordinates of BMY_HPP5 according to Table X,or a mutant or homologue of said molecule or molecular complex.

The invention also provides a method for identifying a mutant ofBMY_HPP5 with altered biological properties, function, or reactivity,the method comprising the step selected from the group consisting of:Using the BMY_HPP5 model or a homologue of said model according to TableX, for the design of protein mutants with altered biological function orproperties.

The invention also provides a method for identifying structural andchemical features of BMY_HPP5 using the structural coordinates set forthin Table X, comprising any steps or combination of steps consisting of:employing identified structural or chemical features to design or selectcompounds as potential BMY_HPP5 modulators; employing thethree-dimensional structural model to design or select compounds aspotential BMY_HPP5 modulators; synthesizing the potential BMY_HPP5modulators; and screening the potential BMY_HPP5 modulators in an assaycharacterized by binding of a protein to the BMY_HPP5. The inventionfurther provides said method wherein the potential BMY_HPP5 modulator isselected from a database. The invention further provides said methodwherein the potential BMY_HPP5 modulator is designed de novo. Theinvention further provides said method wherein the potential BMY_HPP5modulator is designed from a known modulator of activity.

The invention also provides a method for identifying a compound thatmodulates BMY_HPP5 activity, the method comprising any combination ofsteps of: Modeling test compounds that fit spatially into or near theactive site region defined by residues residues D213, H243, C244, andR250 of BMY_HPP5 as defined by structure coordinates according to TableX, or modeling test compounds that fit spatially into athree-dimensional structural model of the catalytic domain of BMY_HPP5,mutant homologue or portion thereof; using said structure coordinates orsaid active site region as set forth in prior claims to identifystructural and chemical features; employing identified structural orchemical features to design or select compounds as potential BMY_HPP5modulators including substrates, antagonists and agonists; employing thethree-dimensional structural model or the catalytic domain of BMY_HPP5to design or select compounds as potential BMY_HPP5 inhibitors;screening the potential BMY_HPP5 inhibitors in an assay characterized bybinding of a test compound to BMY_HPP5; and/or modifying or replacingone or more amino acids from BMY_HPP5 including but not limited to theresidues corresponding to the active site region as set forth in priorclaims of BMY_HPP5 according to Table X.

The invention further relates to a method for preventing, treating, orameliorating a medical condition, wherein the medical condition is arenal condition.

The invention further relates to a method for preventing, treating, orameliorating a medical condition, wherein the medical condition is aninflammatory disease.

The invention further relates to a method for preventing, treating, orameliorating a medical condition, wherein the medical condition is aninflammatory disease where dual-specificity phosphatases, eitherdirectly or indirectly, are involved in disease progression.

The invention further relates to a method for preventing, treating, orameliorating a medical condition, wherein the medical condition is acancer.

The invention further relates to a method for preventing, treating, orameliorating a medical condition, wherein the medical condition is aneural disorder.

The invention further relates to a method for preventing, treating, orameliorating a medical condition, wherein the medical condition is areproductive disorder.

The invention further relates to a method for preventing, treating, orameliorating a medical condition, wherein the medical condition is animmunological disorder.

The invention further relates to a method for preventing, treating, orameliorating a medical condition, wherein the medical condition is amusculo-degenerative disorder.

The invention further relates to a method for preventing, treating, orameliorating a medical condition, wherein the medical condition is amuscle disorder.

The invention further relates to a method for preventing, treating, orameliorating a medical condition, wherein the medical condition is ahepatic disorder.

The invention further relates to a method for preventing, treating, orameliorating a medical condition, wherein the medical condition is anendocrine disorder.

The invention further relates to a method for preventing, treating, orameliorating a medical condition, wherein the medical condition is apulmonary disorder.

The invention further relates to a method for preventing, treating, orameliorating a medical condition, wherein the medical condition is adisorder associated, either directly or indirectly, with TNF-alpha.

The invention further relates to a method for preventing, treating, orameliorating a medical condition, wherein the medical condition is adisorder associated, either directly or indirectly, with IL-1.

BRIEF DESCRIPTION OF THE FIGURES/DRAWINGS

FIG. 1 shows the polynucleotide sequences (SEQ ID NO: 1 and 3) anddeduced amino acid sequence (SEQ ID NO:2 and 4) of gene fragments A andB, respectfully, of the novel human phosphatase, BMY_HPP1, of thepresent invention. The standard one-letter abbreviation for amino acidsis used to illustrate the deduced amino acid sequence. Thepolynucleotide sequence of fragment A contains a sequence of 144nucleotides (SEQ ID NO:1), encoding a polypeptide of 48 amino acids (SEQID NO:2), while the polynucleotide sequence of fragment B contains asequence of 33 nucleotides (SEQ ID NO:3), encoding a polypeptide of 11amino acids (SEQ ID NO:4).

FIG. 2 shows the polynucleotide sequence (SEQ ID NO: 5) and deducedamino acid sequence (SEQ ID NO:6) of a gene fragment of the novel humanphosphatase, BMY_HPP2, of the present invention. The standard one-letterabbreviation for amino acids is used to illustrate the deduced aminoacid sequence. The polynucleotide sequence of this fragment contains asequence of 746 nucleotides (SEQ ID NO:5), encoding 248 amino acids (SEQID NO:6) of the full-length BMY_HPP2 polypeptide, and/or translatedportions of the 5′ and/or 3′ UTR of clone BMY_HPP2. The asterisks (“*”)may represent any amino acid.

FIG. 3 shows the polynucleotide sequence (SEQ ID NO: 7) and deducedamino acid sequence (SEQ ID NO:8) of a gene fragment of the novel humanphosphatase, BMY_HPP3, of the present invention. The standard one-letterabbreviation for amino acids is used to illustrate the deduced aminoacid sequence. The polynucleotide sequence of this fragment contains asequence of 511 nucleotides (SEQ ID NO:5), encoding 170 amino acids (SEQID NO:8) of the full-length BMY_HPP3 polypeptide, and/or translatedportions of the 5′ and/or 3′ UTR of clone BMY_HPP3. The asterisks (“*”)may represent any amino acid.

FIGS. 4A-B show the polynucleotide sequence (SEQ ID NO: 9) and deducedamino acid sequence (SEQ ID NO:10) of a gene fragment of the novel humanphosphatase, BMY_HPP4, of the present invention. The standard one-letterabbreviation for amino acids is used to illustrate the deduced aminoacid sequence. The polynucleotide sequence of this fragment contains asequence of 1710 nucleotides (SEQ ID NO:9), encoding 570 amino acids(SEQ ID NO:10) of the full-length BMY_HPP3 polypeptide, and/ortranslated portions of the 5′ and/or 3′ UTR of clone BMY_HPP4. Theasterisks (“*”) may represent any amino acid.

FIGS. 5A-E show the polynucleotide sequence (SEQ ID NO: 41) and deducedamino acid sequence (SEQ ID NO:42) of the novel full-length humanphosphatase, BMY_HPP5, of the present invention. The standard one-letterabbreviation for amino acids is used to illustrate the deduced aminoacid sequence. The polynucleotide sequence of this protein contains asequence of 5111 nucleotides (SEQ ID NO:41), encoding 665 amino acids(SEQ ID NO:42) of the full-length BMY_HPP5 polypeptide.

FIGS. 6A-D show the regions of identity between the encoded full-lengthhuman phosphatase protein BMY_HPP1 (BMY_HPP1_FL; SEQ ID NO:150), andfragments A and B of BMY_HPP1 (BMY_HPP1_A and BMY_HPP1_B; SEQ ID NO:2and 4, respectfully), to other phosphatase proteins, specifically, theSchizosacchromyces Pombe protein tyrosine phosphatase PYP3 protein(PYP3_SP; Genbank Accession No:gi| P32587; SEQ ID NO:Y7); the mouseprotein tyrosine phosphatase, receptor type, O, protein (MM_RPTPO;Genbank Accession No:gi| NP_(—)035346; SEQ ID NO:Y8); and the humanprotein tyrosine phosphatase, receptor type, O, protein (HS_RPTPO;Genbank Accession No:gi| NP_(—)002839; SEQ ID NO:Y9). The alignment wasperformed using the CLUSTALW algorithm. The darkly shaded amino acidsrepresent regions of matching identity. The lightly shaded amino acidsrepresent regions of matching similarity. Dots (“•”) between residuesindicate gapped regions of non-identity for the aligned polypeptides.Catalytic residues are indicated in bold.

FIGS. 7A-B show the regions of identity between the encoded full-lengthhuman phosphatase protein BMY_HPP2 (BMY_HPP2.FL; SEQ ID NO:152), and thefragment of BMY_HPP2 (BMY_HPP2.partial; SEQ ID NO:6) to otherphosphatase proteins, specifically, the human CDC14 (also known as thecell division cycle 14, S. cerevisiae Gene A protein) homologue A(HS_CDC14A; Genbank Accession No:gi| NP_(—)003663; SEQ ID NO:30); thehuman S. cerevisiae CDC14 homolog, gene B (HS_CDC14B; Genbank AccessionNo:gi| NP_(—)003662; SEQ ID NO:31); and the yeast solubletyrosine-specific protein phosphatase Cdc14p protein (SC_CDC14; GenbankAccession No:gi| NP_(—)002839; SEQ ID NO:32). The alignment wasperformed using the CLUSTALW algorithm. The darkly shaded amino acidsrepresent regions of matching identity. The lightly shaded amino acidsrepresent regions of matching similarity. Dots (“•”) between residuesindicate gapped regions of non-identity for the aligned polypeptides.Catalytic residues are indicated in bold.

FIG. 8 shows the regions of identity between the encoded humanphosphatase protein fragment of BMY_HPP3 (SEQ ID NO:8) to otherphosphatase proteins, specifically, the human protein tyrosinephosphatase PTPCAAX1 PROTEIN (HS_PTPCAAX1; Genbank Accession No:gi|AAB40597; SEQ ID NO:33); the human protein tyrosine phosphatase PTPCAAX2(HS_PTPCAAX2; Genbank Accession No:gi| AAB40598; SEQ ID NO:34); themouse prenylated protein tyrosine phosphatase (MM_PTPCAAX; GenbankAccession No:gi| JC5981; SEQ ID NO:35); and the Drosophila PRL-1 protein(DM_PRL1; Genbank Accession No:gi| AAF53506; SEQ ID NO:36). Thealignment was performed using the CLUSTALW algorithm. The darkly shadedamino acids represent regions of matching identity. The lightly shadedamino acids represent regions of matching similarity. Dots (“•”) betweenresidues indicate gapped regions of non-identity for the alignedpolypeptides. Catalytic residues are indicated in bold.

FIGS. 9A-B show the regions of identity between the encoded humanphosphatase protein fragment of BMY_HPP4 (SEQ ID NO:10) to otherphosphatase proteins, specifically, the mouse osteotesticular proteintyrosine phosphatase (MM_OST-PTP; Genbank Accession No:gi| AAG28768; SEQID NO:37); and the rat protein-tyrosine-phosphatase (RN_PTP-OST; GenbankAccession No:gi| A55148; SEQ ID NO:38). The alignment was performedusing the CLUSTALW algorithm. The darkly shaded amino acids representregions of matching identity. The lightly shaded amino acids representregions of matching similarity. Dots (“•”) between residues indicategapped regions of non-identity for the aligned polypeptides. Catalyticresidues are indicated in bold.

FIGS. 10A-B shows the regions of identity between the encoded humanphosphatase protein fragment of BMY_HPP5 (SEQ ID NO:42) to otherphosphatase proteins, specifically, the human dual specificityphosphatase 8 (hs_dspp8; Genbank Accession No:gi| NP_(—)004411; SEQ IDNO:39); and the mouse neuronal tyrosine/threonine phosphatase 1 (rmm_npp1; Genbank Accession No:gi| NP_(—)032774; SEQ ID NO:40). Thealignment was performed using the CLUSTALW algorithm. The darkly shadedamino acids represent regions of matching identity. The lightly shadedamino acids represent regions of matching similarity. Dots (“•”) betweenresidues indicate gapped regions of non-identity for the alignedpolypeptides. Catalytic residues are indicated in bold.

FIG. 11 shows an expression profile of the novel human phosphataseprotein BMY_HPP5. The figure illustrates the relative expression levelof BMY_HPP5 amongst various mRNA tissue sources. As shown, the BMY_HPP5polypeptide was expressed to a significant extent, in the testis andspinal cord, and to a lesser extent, in bone marrow, brain, liver, andthymus. Expression data was obtained by measuring the steady stateBMY_HPP5 mRNA levels by quantitative PCR using the PCR primer pairprovided as SEQ ID NO:67 and 68 as described herein.

FIG. 12 shows a table illustrating the percent identity and percentsimilarity between the BMY_HPP5 (SEQ ID NO:42), the human RET31 (SEQ IDNO:109), and the mouse RET31 (SEQ ID NO:114) polypeptides of the presentinvention with other phosphatase proteins. The percent identity andpercent similarity values were determined based upon the GAP algorithm(GCG suite of programs; and Henikoff, S. and Henikoff, J. G., Proc.Natl. Acad. Sci. USA 89: 10915-10919 (1992)) using the followingparameters: gap weight=8, and length weight=2.

FIGS. 13A-F show the polynucleotide sequence (SEQ ID NO: 108) anddeduced amino acid sequence (SEQ ID NO:109) of the novel full-lengthhuman phosphatase, RET31, of the present invention. The standardone-letter abbreviation for amino acids is used to illustrate thededuced amino acid sequence. The polynucleotide sequence of this proteincontains a sequence of 5450 nucleotides (SEQ ID NO:108), encoding 665amino acids (SEQ ID NO:109) of the full-length RET31 polypeptide. Ananalysis of the RET31 polypeptide determined that it comprised thefollowing features: a dual specificity phosphatase catalytic domainlocated from about amino acid 158 to about amino acid 297 (SEQ IDNO:134) of SEQ ID NO:109 represented by double underlining; and acatalytic cysteine amino acid residue located at amino acid 244 of SEQID NO:109 represented by shading.

FIGS. 14A-C show the regions of identity between the encoded humanphosphatase protein of RET31 (SEQ ID NO:109) to other phosphataseproteins, specifically, the human protein-tyrosine phosphatase DUS8protein, also referred to as hVH-5 (DUS8; Genbank AccessionNo:gi|U27193; SEQ ID NO:110); the human dual specificity MAP kinaseDUSP6 protein (DUSP6; Genbank Accession No:gi|AB013382; SEQ ID NO:111);the human map kinase phosphatase MKP-5 protein (MKP-5; Genbank AccessionNo:gi|AB026436; SEQ ID NO:112); and the mouse RET31 protein of thepresent invention (mRET31; SEQ ID NO:114). The alignment was performedusing the CLUSTALW algorithm. The darkly shaded amino acids representregions of matching identity. The lightly shaded amino acids representregions of matching similarity. Dots (“•”) between residues indicategapped regions of non-identity for the aligned polypeptides.

FIG. 15 shows the results of a northern hybridization illustrating theexpression profile of the novel human phosphatase protein RET31. Thefigure illustrates the relative expression level of RET31 amongstvarious mRNA tissue sources. As shown, the RET31 polypeptide wasexpressed predominately in adrenal gland, testis, and skeletal muscle;to a significant extent, in the liver, prostate ovary, and to a lesserextent, in placenta, pancreas, thymus, small intestine, thyroid, heart,kidney and liver. Expression data was obtained by the hybridization of a408 bp P³²-labeled RET31 polynucleotide fragment correponding to SEQ IDNO:108 (specifically the RsaI fragment of SEQ ID NO:115) to severalmultiple tissue northern mRNA blots as described herein.

FIGS. 16A-C show the polynucleotide sequence (SEQ ID NO: 113) anddeduced amino acid sequence (SEQ ID NO:114) of the novel full-lengthmouse phosphatase, mRET31, of the present invention. The standardone-letter abbreviation for amino acids is used to illustrate thededuced amino acid sequence. The polynucleotide sequence of this proteincontains a sequence of 2756 nucleotides (SEQ ID NO:113), encoding 660amino acids (SEQ ID NO:114) of the full-length mRET31 polypeptide. Ananalysis of the mRET31 polypeptide determined that it comprised thefollowing features: a dual specificity phosphatase catalytic domainlocated from about amino acid 158 to about amino acid 297 (SEQ IDNO:135) of SEQ ID NO:114 represented by double underlining.

FIG. 17 shows the regions of identity between the dual specificityphosphatase catalytic (DSPc) domain of the encoded human phosphataseprotein of RET31 (SEQ ID NO:109) to the dual specificity phosphatasecatalytic (DSPc) domain of other phosphatase proteins, specifically, theDSPc domain of the human protein-tyrosine phosphatase DUS8 protein, alsoreferred to as hVH-5 (DUS8_DSPc; Genbank Accession No:gi|U27193; SEQ IDNO:110); the DSPc domain of the human dual specificity MAP kinase DUSP6protein (DUSP6_DSPc; Genbank Accession No:gi|AB013382; SEQ ID NO:111);and the DSPc domain of the human map kinase phosphatase MKP-5 protein(MKP-5_DSPc; Genbank Accession No:gi|AB026436; SEQ ID NO:112. Red boxesindicate conservation among all four DSPc domains, blue boxes indicateconservation among three DSPc domains, and green boxes indicateconservation between RET31 and one of the other protein domains. Dots(“•”) between residues indicate gapped regions of non-identity for thealigned polypeptides.

FIG. 18 shows the results of a northern hybridization illustrating theexpression profile of the novel human phosphatase protein RET31 in humanlung microvascular endothelial cells (HMCEC) after the administration ofTNF-alpha for 0, 1, 6, and 24 hours. As shown, the RET31 polypeptide isup-regulated by TNF-α, reaching a peak of expression of about 6 hours.Expression data was obtained by the hybridization of a 408 bpP³²-labeled RET31 polynucleotide fragment correponding to SEQ ID NO:108(specifically the RsaI fragment of SEQ ID NO:115) to northern blotscontaining the isolated HMVEC mRNA for each indicated sample asdescribed herein.

FIGS. 19A-F show the predicted polynucleotide sequence (SEQ ID NO:147)and deduced amino acid sequence (SEQ ID NO:148) of the novel full-lengthhuman phosphatase, RET31, of the present invention. The standardone-letter abbreviation for amino acids is used to illustrate thededuced amino acid sequence. The polynucleotide sequence of this proteincontains a sequence of 5450 nucleotides (SEQ ID NO:147), encoding 665amino acids (SEQ ID NO:148) of the full-length RET31 polypeptide. Aportion of the sequence was determined based upon the sequence providedfrom the Incyte gene cluster 1026659.7 using bioinformatic methods.

FIGS. 20A-D show the predicted polynucleotide sequence (SEQ ID NO:149)and deduced amino acid sequence (SEQ ID NO:150) of the novel full-lengthhuman phosphatase, BMY_HPP1, of the present invention. The standardone-letter abbreviation for amino acids is used to illustrate thededuced amino acid sequence. The polynucleotide sequence of this proteincontains a sequence of 4393 nucleotides (SEQ ID NO:149), encoding 607amino acids (SEQ ID NO:150) of the full-length BMY_HPP1 polypeptide. Ananalysis of the BMY_HPP1 polypeptide determined that it comprised thefollowing features: a predicted dual specificity phosphatase catalyticdomain located from about amino acid 41 to about amino acid 49 of SEQ IDNO:150 represented by shading; and conserved phophatase catalyticresidues at amino acid 14, at amino acid 42, and at amino acid 48 of SEQID NO:150 (FIGS. 20A-D).

FIG. 21 shows the polynucleotide sequence (SEQ ID NO:151) and deducedamino acid sequence (SEQ ID NO:152) of the novel full-length humanphosphatase, BMY_HPP2, of the present invention. The standard one-letterabbreviation for amino acids is used to illustrate the deduced aminoacid sequence. The polynucleotide sequence of this protein contains asequence of 878 nucleotides (SEQ ID NO:151), encoding 150 amino acids(SEQ ID NO:152) of the full-length BMY_HPP2 polypeptide. An analysis ofthe BMY_HPP2 polypeptide determined that it comprised the followingfeatures: a predicted dual specificity phosphatase catalytic domainlocated from about amino acid 93 and 94, and from about amino acid 100and 101 of SEQ ID NO:152 represented by shading; and conservedphosphatase catalytic residues located at amino acid 65, 94, and 100 ofSEQ ID NO:152 represented in bold.

FIG. 22 shows an expression profile of the novel full-length humanphosphatase protein BMY_HPP1. The figure illustrates the relativeexpression level of BMY_HPP1 amongst various mRNA tissue sources. Asshown, the BMY_HPP1 polypeptide was expressed predominately in testis;to a significant extent, in the spinal cord, and to a lesser extent, inpancreas, brain, pituitary, heart, and lung. Expression data wasobtained by measuring the steady state BMY_HPP1 mRNA levels byquantitative PCR using the PCR primer pair provided as SEQ ID NO:154 and155 as described herein.

FIG. 23 shows an expression profile of the novel full-length humanphosphatase protein BMY_HPP2. The figure illustrates the relativeexpression level of BMY_HPP2 amongst various mRNA tissue sources. Asshown, the BMY_HPP2 polypeptide was expressed predominately in liver andkidney; to a significant extent, in the spleen, and to a lesser extent,in lung, testis, heart, intestine, pancreas, lymph node, spinal cord,and prostate. Expression data was obtained by measuring the steady stateBMY_HPP2 mRNA levels by quantitative PCR using the PCR primer pairprovided as SEQ ID NO:156 and 157 as described herein.

FIG. 24 shows a table illustrating the percent identity and percentsimilarity between the full-length BMY_HPP1 polypeptide (SEQ ID NO:150),and the full-length BMY_HPP2 polypeptide (SEQ ID NO:152) of the presentinvention with other phosphatase proteins. The percent identity andpercent similarity values were determined based upon the GAP algorithm(GCG suite of programs; and Henikoff, and Henikoff, J. G., Proc. Natl.Acad. Sci. USA 89: 10915-10919 (1992)) using the following parameters:gap weight=8, and length weight=2.

FIG. 25 shows a table illustrating the percent identity and percentsimilarity between the full-length RET31 polypeptide (SEQ ID NO:109) ofthe present invention with other phosphatase proteins. The percentidentity and percent similarity values were determined based upon theGAP algorithm (GCG suite of programs; and Henikoff, and Henikoff, J. G.,Proc. Natl. Acad. Sci. USA 89: 10915-10919 (1992)) using the followingparameters: gap weight=8, and length weight=2.

FIG. 26 shows an expanded expression profile of the novel full-lengthhuman phosphatase protein BMY_HPP1. The figure illustrates the relativeexpression level of BMY_HPP1 amongst various mRNA tissue sources. Asshown, the BMY_HPP1 polypeptide was expressed predominately in brainsubregions and other central nervous system tissues, in particular thecaudate, hippocampus and nucleus accumbens of the brain. Significantexpression was observed in the in the adrenal, pineal and pituitaryglands, the atrium of the heart, in the testis, and to a lesser extentin a number of other tissues as shown. Expression data was obtained bymeasuring the steady state BMY_HPP1 mRNA levels by quantitative PCRusing the PCR primer pair provided as SEQ ID NO:194 and 195, and Taqmanprobe (SEQ ID NO:196) as described in Example 59 herein.

FIG. 27 shows the regions of identity between amino acid residues M1 toE301 of the BMY_HPP1 polypeptide (amino acids M1 to E301 of SEQ IDNO:150) to amino acid residues D11 to N321 of the human tyrosinespecific phosphatase 1aax (Protein Data Bank, PDB entry 1aax chain A;Genbank Accession No. gi|2981942; SEQ ID NO:206) which was used as thebasis for building the BMY_HPP1 homology model as represented in TableVIII and visualized in FIG. 28. Amino acids defining active siteresidues are highlighted with asterisks (“*”). The alignment was createdusing the FASTA algorithm (Pearson, et. al. 1990).

FIG. 28 shows a three-dimensional homology model of amino acid residuesM1 to E301 of the BMY_HPP1 polypeptide based upon the homologousstructure of amino acid residues D11 to N321 of the human tyrosinespecific phosphatase 1aax (Protein Data Bank, PDB entry 1aax chain A;Genbank Accession No. gi|2981942; SEQ ID NO:206). The structuralcoordinates of the BMY_HPP1 polypeptide are provided in Table VIIIherein. The homology model of BMY_HPP1 was derived from generating asequence alignment with the the human tyrosine specific phosphatase 1aax(Protein Data Bank, PDB entry 1aax chain A; Genbank Accession No.gi|2981942; SEQ ID NO:206) using the INSIGHTII (Accelrys Inc., SanDiego, Calif.) version 2000 as described herein.

FIG. 29 shows an energy graph for the BMY_HPP1 model of the presentinvention (dotted line) and the tyrosine specific phosphatase 1aaxtemplate (solid line) from which the model was generated. The energydistribution for each protein fold is displayed on the y-axis, while theamino acid residue position of the protein fold is displayed on thex-axis. As shown, the BMY_HPP1 model has slightly higher energies in theC-terminal region while the N-terminal region of the structural modelappears to represent a “native-like” conformation of the BMY_HPP1polypeptide. This graph supports the motif and sequence alignments inconfirming that the three dimensional structure coordinates of BMY_HPP1are an accurate and useful representation of the structure of theBMY_HPP1 polypeptide.

FIG. 30 shows an expanded expression profile of the novel full-lengthhuman phosphatase protein BMY_HPP2. The figure illustrates the relativeexpression level of BMY_HPP2 amongst various mRNA tissue sources. Asshown, the BMY_HPP2 polypeptide was expressed predominately in adrenalgland; significantly in the pineal and pituitary gland, lung parenchyma,bronchi, kidney, liver, blood vessels from the choroid plexus, coronaryartery, pulmonary artery, the nucleus accumbens of the brain, and to alesser extent in the trachea, breast and uterus and in other tissues asshown. Expression data was obtained by measuring the steady stateBMY_HPP2 mRNA levels by quantitative PCR using the PCR primer pairprovided as SEQ ID NO:197 and 198, and Taqman probe (SEQ ID NO:199) asdescribed in Example 59 herein.

FIG. 31 shows the regions of identity between amino acid residues M1 toK150 of the BMY_HPP2 polypeptide (amino acids M1 to K150 of SEQ IDNO:152) to amino acid residues N31 to K179 of the N-terminus of thehuman dual specificity phosphatase, 1vhr (vaccinia H1-relatedphosphatase VN1) (residues N31-K179; Protein Data Bank, PDB entry 1vhrchain A; Genbank Accession No. gi|1633321; SEQ ID NO:207) which was usedas the basis for building the BMY_HPP2 homology model as represented inTable IX and visualized in FIG. 32. Amino acids defining active siteresidues are highlighted in bold. The alignment was created using theFASTA algorithm (Pearson, et. al. 1990).

FIG. 32 shows a three-dimensional homology model of amino acid residuesM1 to K150 of the BMY_HPP2 polypeptide based upon the homologousstructure of amino acid residues N31 to K179 of the N-terminus of thehuman dual specificity phosphatase, 1vhr (vaccinia H1-relatedphosphatase VN1) (residues N31-K179; Protein Data Bank, PDB entry 1vhrchain A; Genbank Accession No. gi|1633321; SEQ ID NO:207). Thestructural coordinates of the BMY_HPP2 polypeptide are provided in TableIX herein. The homology model of BMY_HPP2 was derived from generating asequence alignment with the human dual specificity phosphatase, 1vhr(vaccinia H1-related phosphatase VN1) (residues N31-K179; Protein DataBank, PDB entry 1vhr chain A; Genbank Accession No. gi|1633321; SEQ IDNO:207) using the INSIGHTII (Accelrys Inc., San Diego, Calif.) version2000 as described herein.

FIG. 33 shows an energy graph for the BMY_HPP2 model of the presentinvention (dotted line) and the phosphatase VHR template (PDB code 1vhr)(solid line) from which the model was generated. The energy distributionfor each protein fold is displayed on the y-axis, while the amino acidresidue position of the protein fold is displayed on the x-axis. Asshown, the BMY_HPP2 model and 1vhr template have similar energies overthe aligned region, suggesting that the structural model of BMY_HPP2represents a “native-like” conformation of the BMY_HPP2 polypeptide.This graph supports the motif and sequence alignments in confirming thatthe three dimensional structure coordinates of BMY_HPP2 are an accurateand useful representation of the structure of the BMY_HPP1 polypeptide.

FIG. 34 shows an expanded expression profile of the novel full-lengthhuman phosphatase protein BMY_HPP4. The figure illustrates the relativeexpression level of BMY_HPP4 amongst various mRNA tissue sources. Asshown, the BMY_HPP4 polypeptide was expressed predominately incerebellum; significantly in other subregions of the brain, and in thepineal and pituitary glands. Expression data was obtained by measuringthe steady state BMY_HPP4 mRNA levels by quantitative PCR using the PCRprimer pair provided as SEQ ID NO:200 and 201, and Taqman probe (SEQ IDNO:202) as described in Example 59 herein.

FIG. 35 shows an expanded expression profile of the novel full-lengthhuman phosphatase protein BMY_HPP5. The figure illustrates the relativeexpression level of BMY_HPP5 amongst various mRNA tissue sources. Asshown, the BMY_HPP5 polypeptide was expressed predominately in theadrenal, pineal and pituitary glands; significantly in the cerebellum,prostate, testis, and to a lesser extent in other tissues as shown.Expression data was obtained by measuring the steady state BMY_HPP5 mRNAlevels by quantitative PCR using the PCR primer pair provided as SEQ IDNO:203 and 204, and Taqman probe (SEQ ID NO:205) as described in Example59 herein.

FIG. 36 shows the results of para-nitrophenylphosphate (pNPP)phosphatase activity assays of the purified RET31-GST full length (FL),and M1 to T302 RET31 C-terminal deletion mutant (trunc) fusion proteins,as compared to purified GST alone. The bars represent the average oftriplicate determinations, and the standard deviations are as shown.Each protein preparation was assayed in the absence and presence of 2 mMorthovanadate (”-van”). As shown, both the full-length RET31 and M1 toT302 RET31 C-terminal deletion mutant demonstrated phosphatase activityvia cleavage of the NPP substrate which was blocked by thephosphatase-specific inhibitor, vanadate. Of particular significance isthe unexpected five fold increase in phosphatase activity of the M1 toT302 RET31 C-terminal deletion mutant relative to the full-length RET31polypeptide. The phosphatase assays were performed as described inExample 57 herein. The full length and truncated versions clearlydemonstrated phosphatase activity compared to the GST protein.

FIG. 37 shows the regions of identity between amino acid residues N157to 1300 of the BMY_HPP5 polypeptide (amino acids N157 to 1300 of SEQ IDNO:42) to amino acid residues A204 to L347 of the human dual specificityphosphatase MAP Kinase phosphatase 3, also called PYST1, 1 mkp (residuesA204-L347; Protein Data Bank, PDB entry 1 mkp chain A; Genbank AccessionNo. gi|5822131; SEQ ID NO:208) which was used as the basis for buildingthe BMY_HPP5 homology model as represented in Table X and visualized inFIG. 38. Amino acids defining active site residues are highlighted inbold. The alignment was created using the FASTA algorithm (Pearson, et.al. 1990).

FIG. 38 shows a three-dimensional homology model of amino acid residuesN157 to 1300 of the BMY_HPP5 polypeptide based upon the homologousstructure of amino acid residues A204 to L347 of the human dualspecificity phosphatase MAP Kinase phosphatase 3, also called PYST1, 1mkp (residues A204-L347; Protein Data Bank, PDB entry 1 mkp chain A;Genbank Accession No. gi|5822131; SEQ ID NO:208). The structuralcoordinates of the BMY_HPP2 polypeptide are provided in Table IX herein.The homology model of BMY_HPP2 was derived from generating a sequencealignment with the human dual specificity phosphatase MAP Kinasephosphatase 3, also called PYST1, 1 mkp (residues A204-L347; ProteinData Bank, PDB entry 1 mkp chain A; Genbank Accession No. gi|5822131;SEQ ID NO:208) using the INSIGHTII (Accelrys Inc., San Diego, Calif.)version 2000 as described herein.

FIG. 39 shows an energy graph for the BMY_HPP5 model of the presentinvention (dotted line) and the phosphatase VHR template (PDB code 1vhr)(solid line) from which the model was generated. The energy distributionfor each protein fold is displayed on the y-axis, while the amino acidresidue position of the protein fold is displayed on the x-axis. Asshown, the BMY_HPP5 model and 1vhr template have similar energies overthe aligned region, suggesting that the structural model of BMY_HPP5represents a “native-like” conformation of the BMY_HPP5 polypeptide.This graph supports the motif and sequence alignments in confirming thatthe three dimensional structure coordinates of BMY_HPP5 are an accurateand useful representation of the structure of the BMY_HPP5 polypeptide.

Table I provides a summary of the novel polypeptides and their encodingpolynucleotides of the present invention.

Table II illustrates the preferred hybridization conditions for thepolynucleotides of the present invention. Other hybridization conditionsmay be known in the art or are described elsewhere herein.

Table III provides the amino acid sequences of known phosphatases thatwere used to identify the novel human phosphatases of the presentinvention using the BLAST algorithm as described herein.

Table IV provides the PFAM motifs that were used in Hidden Markov Model(HMM) searches to identify the novel human phosphtases of the presentinvention as described herein.

Table V provides the predicted exon structure of the BMY_HPP4 gene. The‘Start’ and ‘End’ designations refer to the respective nucleotidepositions of the BMY_HPP4 as they appear for the corresponding genomicsequence in BAC AL 354751. The numbering begins at the start of BACAL354751; nucleotide 71352 in the BAC is equivalent to nucleotide 1 ofthe BMY_HPP4 transcript (SEQ ID NO:9; FIG. 4).

Table VI provides representative primers for sequencing and/or cloningany one of the human phosphatases of the present invention inconjunction with the teachings described herein. ‘Left Cloning Primer’,and ‘Right Cloning Primer’ represent the forward and reverse sequencingprimers, while the ‘Internal RevComp Cloning Primer’ and/or ‘InternalCloning Primer’ represent antisense cloning primers as described in theExamples herein.

Table VII provides a summary of various conservative substitutionsencompassed by the present invention.

Table VIII provides the structural coordinates of the homology model ofthe BMY_HPP1 polypeptide provided in FIG. 28. A description of theheadings are as follows: “Atom No” refers to the atom number within theBMY_HPP1 homology model; “Atom name” refers to the element whosecoordinates are measured, the first letter in the column defines theelement; “Residue” refers to the amino acid of the BMY_HPP1 polypeptidewithin which the atom resides; “Residue No” refers to the amino acidposition in which the atom resides, “X Coord”, “Y Coord”, and “Z Coord”structurally define the atomic position of the element measured in threedimensions.

Table IX provides the structural coordinates of the homology model ofthe BMY_HPP2 polypeptide provided in FIG. 32. A description of theheadings are as follows: “Atom No” refers to the atom number within theBMY_HPP2 homology model; “Atom name” refers to the element whosecoordinates are measured, the first letter in the column defines theelement; “Residue” refers to the amino acid of the BMY_HPP2 polypeptidewithin which the atom resides; “Residue No” refers to the amino acidposition in which the atom resides, “X Coord”, “Y Coord”, and “Z Coord”structurally define the atomic position of the element measured in threedimensions.

Table X provides the structural coordinates of the homology model of theBMY_HPP5 polypeptide provided in FIG. 38. A description of the headingsare as follows: “Atom No” refers to the atom number within the BMY_HPP5homology model; “Atom name” refers to the element whose coordinates aremeasured, the first letter in the column defines the element; “Residue”refers to the amino acid of the BMY_HPP5 polypeptide within which theatom resides; “Residue No” refers to the amino acid position in whichthe atom resides, “X Coord”, “Y Coord”, and “Z Coord” structurallydefine the atomic position of the element measured in three dimensions.

DETAILED DESCRIPTION OF THE INVENTION

The present invention may be understood more readily by reference to thefollowing detailed description of the preferred embodiments of theinvention and the Examples included herein. All references to“phosphatase” and/or “human phosphatases” shall be construed to apply toBMY_HPP1, BMY_HPP2, BMY_HPP3, BMY_HPP4, BMY_HPP5, RET31, mouse RET31,and/or fragments thereof unless otherwise specified herein. Moreover,since BMY_HPP5 is believed to represent a splice variant of the RET31polypeptide, all references to “BMY_HPP5” shall be construed to apply toRET31, and all references to “RET31” shall be construed to apply to“BMY_HPP5”.

The invention provides human polynucleotide sequences encoding novelhuman phosphatases with substantial homology to the class ofphosphatases known as phosphotyrosine or dual-specificity (P-Tyr, P-Serand P-Thr) phosphatases. Members of this class of phosphatases have beenimplicated in a number of diseases and/or disorders, which include, butare not limited to, bone disorders, (Yoon, H K., Baylink, D J., Lau, KH, Am. J. Nephrol., 20(2):153-62, (2000)), disease resistance topathogens, reproductive disorders (Gloria, Bottini, F., Nicotra, M.,Lucarini, N., Borgiani, P., La, Torre, M., Amante, A., Gimelfarb, A.,Bottini, E, Dis. Markers., 12(4):261-9, (1996)), neural disorders(Shimohama, S., Fujimoto, S., Taniguchi, T., Kameyama, M., Kimura, J.Ann, Neurol., 33(6):616-21, (1993)), prostate cancer (Nguyen, L.,Chapdelaine, A., and Chevalier, S., Clin. Chem. 36(8 Pt 1): 1450-5(1990)), immune disorders, particularly those relating to haematopoieticcell development, apoptosis, activation, and nonresponsiveness(Frearson, J A., Alexander, D R, Bioessays., 19(5): 417-27 (1997)), etc.

In the present invention, “isolated” refers to material removed from itsoriginal environment (e.g., the natural environment if it is naturallyoccurring), and thus is altered “by the hand of man” from its naturalstate. For example, an isolated polynucleotide could be part of a vectoror a composition of matter, or could be contained within a cell, andstill be “isolated” because that vector, composition of matter, orparticular cell is not the original environment of the polynucleotide.The term “isolated” does not refer to genomic or cDNA libraries, wholecell total or mRNA preparations, genomic DNA preparations (includingthose separated by electrophoresis and transferred onto blots), shearedwhole cell genomic DNA preparations or other compositions where the artdemonstrates no distinguishing features of the polynucleotide/sequencesof the present invention.

In specific embodiments, the polynucleotides of the invention are atleast 15, at least 30, at least 50, at least 100, at least 125, at least500, or at least 1000 continuous nucleotides but are less than or equalto 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, 7.5 kb, 5 kb, 2.5 kb,2.0 kb, or 1 kb, in length. In a further embodiment, polynucleotides ofthe invention comprise a portion of the coding sequences, as disclosedherein, but do not comprise all or a portion of any intron. In anotherembodiment, the polynucleotides comprising coding sequences do notcontain coding sequences of a genomic flanking gene (i.e., 5′ or 3′ tothe gene of interest in the genome). In other embodiments, thepolynucleotides of the invention do not contain the coding sequence ofmore than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1genomic flanking gene(s).

As used herein, a “polynucleotide” refers to a molecule having a nucleicacid sequence contained in SEQ ID NO:7, 9, 41, 108, 149, 151 or the cDNAcontained within the clone deposited with the ATCC. For example, thepolynucleotide can contain the nucleotide sequence of the full lengthcDNA sequence, including the 5′ and 3′ untranslated sequences, thecoding region, with or without a signal sequence, the secreted proteincoding region, as well as fragments, epitopes, domains, and variants ofthe nucleic acid sequence. Moreover, as used herein, a “polypeptide”refers to a molecule having the translated amino acid sequence generatedfrom the polynucleotide as broadly defined.

In the present invention, the full length sequence identified as SEQ IDNO: 7, 9, 41, 108, 149, 151 was often generated by overlapping sequencescontained in one or more clones (contig analysis). A representativeclone containing all or most of the sequence for SEQ ID NO:X wasdeposited with the American Type Culture Collection (“ATCC”). As shownin Table I, each clone is identified by a cDNA Clone ID (Identifier) andthe ATCC Deposit Number. The ATCC is located at 10801 UniversityBoulevard, Manassas, Va. 20110-2209, USA. The ATCC deposit was madepursuant to the terms of the Budapest Treaty on the internationalrecognition of the deposit of microorganisms for purposes of patentprocedure. The deposited clone is inserted in the pSport plasmid (LifeTechnologies) using SalI and NotI restriction sites as described herein.

Unless otherwise indicated, all nucleotide sequences determined bysequencing a DNA molecule herein were determined using an automated DNAsequencer (such as the Model 373 from Applied Biosystems, Inc.), and allamino acid sequences of polypeptides encoded by DNA molecules determinedherein were predicted by translation of a DNA sequence determined above.Therefore, as is known in the art for any DNA sequence determined bythis automated approach, any nucleotide sequence determined herein maycontain some errors. Nucleotide sequences determined by automation aretypically at least about 90% identical, more typically at least about95% to at least about 99.9% identical to the actual nucleotide sequenceof the sequenced DNA molecule. The actual sequence can be more preciselydetermined by other approaches including manual DNA sequencing methodswell known in the art. As is also known in the art, a single insertionor deletion in a determined nucleotide sequence compared to the actualsequence will cause a frame shift in translation of the nucleotidesequence such that the predicted amino acid sequence encoded by adetermined nucleotide sequence will be completely different from theamino acid sequence actually encoded by the sequenced DNA molecule,beginning at the point of such an insertion or deletion.

Using the information provided herein, such as the nucleotide sequenceprovided as SEQ ID NO: 7, 9, 41, 108, 149, 151, a nucleic acid moleculeof the present invention encoding a human phosphatase polypeptide may beobtained using standard cloning and screening procedures, such as thosefor cloning cDNAs using mRNA as starting material.

A “polynucleotide” of the present invention also includes thosepolynucleotides capable of hybridizing, under stringent hybridizationconditions, to sequences contained in SEQ ID NO:X, the complementthereof, or the cDNA within the clone deposited with the ATCC.“Stringent hybridization conditions” refers to an overnight incubationat 42 degree C. in a solution comprising 50% formamide, 5×SSC (750 mMNaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured,sheared salmon sperm DNA, followed by washing the filters in 0.1×SSC atabout 65 degree C.

Also contemplated are nucleic acid molecules that hybridize to thepolynucleotides of the present invention at lower stringencyhybridization conditions. Changes in the stringency of hybridization andsignal detection are primarily accomplished through the manipulation offormamide concentration (lower percentages of formamide result inlowered stringency); salt conditions, or temperature. For example, lowerstringency conditions include an overnight incubation at 37 degree C. ina solution comprising 6×SSPE (20×SSPE=3M NaCl; 0.2M NaH2PO4; 0.02M EDTA,pH 7.4), 0.5% SDS, 30% formamide, 100 ug/ml salmon sperm blocking DNA;followed by washes at 50 degree C. with 1×SSPE, 0.1% SDS. In addition,to achieve even lower stringency, washes performed following stringenthybridization can be done at higher salt concentrations (e.g. 5×SSC).

Note that variations in the above conditions may be accomplished throughthe inclusion and/or substitution of alternate blocking reagents used tosuppress background in hybridization experiments. Typical blockingreagents include Denhardt's reagent, BLOTTO, heparin, denatured salmonsperm DNA, and commercially available proprietary formulations. Theinclusion of specific blocking reagents may require modification of thehybridization conditions described above, due to problems withcompatibility.

Of course, a polynucleotide which hybridizes only to polyA+ sequences(such as any 3′ terminal polyA+ tract of a cDNA shown in the sequencelisting), or to a complementary stretch of T (or U) residues, would notbe included in the definition of “polynucleotide,” since such apolynucleotide would hybridize to any nucleic acid molecule containing apoly (A) stretch or the complement thereof (e.g., practically anydouble-stranded cDNA clone generated using oligo dT as a primer).

The polynucleotide of the present invention can be composed of anypolyribonucleotide or polydeoxribonucleotide, which may be unmodifiedRNA or DNA or modified RNA or DNA. For example, polynucleotides can becomposed of single- and double-stranded DNA, DNA that is a mixture ofsingle- and double-stranded regions, single- and double-stranded RNA,and RNA that is mixture of single- and double-stranded regions, hybridmolecules comprising DNA and RNA that may be single-stranded or, moretypically, double-stranded or a mixture of single- and double-strandedregions. In addition, the polynucleotide can be composed oftriple-stranded regions comprising RNA or DNA or both RNA and DNA. Apolynucleotide may also contain one or more modified bases or DNA or RNAbackbones modified for stability or for other reasons. “Modified” basesinclude, for example, tritylated bases and unusual bases such asinosine. A variety of modifications can be made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically, or metabolicallymodified forms.

The polypeptide of the present invention can be composed of amino acidsjoined to each other by peptide bonds or modified peptide bonds, i.e.,peptide isosteres, and may contain amino acids other than the 20gene-encoded amino acids. The polypeptides may be modified by eithernatural processes, such as posttranslational processing, or by chemicalmodification techniques which are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.It will be appreciated that the same type of modification may be presentin the same or varying degrees at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Polypeptides may be branched, for example, as a result ofubiquitination, and they may be cyclic, with or without branching.Cyclic, branched, and branched cyclic polypeptides may result fromposttranslation natural processes or may be made by synthetic methods.Modifications include acetylation, acylation, ADP-ribosylation,amidation, covalent attachment of flavin, covalent attachment of a hememoiety, covalent attachment of a nucleotide or nucleotide derivative,covalent attachment of a lipid or lipid derivative, covalent attachmentof phosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cysteine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, pegylation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination. (See, forinstance, PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993); POSTTRANSLATIONALCOVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press,New York, pgs. 1-12 (1983); Seifter et al., Meth Enzymol 182:626-646(1990); Rattan et al., Ann NY Acad Sci 663:48-62 (1992).)

“SEQ ID NO:X” refers to a polynucleotide sequence while “SEQ ID NO:Y”refers to a polypeptide sequence, both sequences are identified by aninteger specified in Table I.

“A polypeptide having biological activity” refers to polypeptidesexhibiting activity similar, but not necessarily identical to, anactivity of a polypeptide of the present invention, including matureforms, as measured in a particular biological assay, with or withoutdose dependency. In the case where dose dependency does exist, it neednot be identical to that of the polypeptide, but rather substantiallysimilar to the dose-dependence in a given activity as compared to thepolypeptide of the present invention (i.e., the candidate polypeptidewill exhibit greater activity or not more than about 25-fold less and,preferably, not more than about tenfold less activity, and mostpreferably, not more than about three-fold less activity relative to thepolypeptide of the present invention.)

The term “organism” as referred to herein is meant to encompass anyorganism referenced herein, though preferably to eukaryotic organsisms,more preferably to mammals, and most preferably to humans.

The present invention encompasses the identification of proteins,nucleic acids, or other molecules, that bind to polypeptides andpolynucleotides of the present invention (for example, in areceptor-ligand interaction). The polynucleotides of the presentinvention can also be used in interaction trap assays (such as, forexample, that described by Ozenberger and Young (Mol Endocrinol.,9(10):1321-9, (1995); and Ann. N.Y. Acad. Sci., 7; 766:279-81, (1995)).

The polynucleotide and polypeptides of the present invention are usefulas probes for the identification and isolation of full-length cDNAsand/or genomic DNA which correspond to the polynucleotides of thepresent invention, as probes to hybridize and discover novel, relatedDNA sequences, as probes for positional cloning of this or a relatedsequence, as probe to “subtract-out” known sequences in the process ofdiscovering other novel polynucleotides, as probes to quantify geneexpression, and as probes for microarrays.

In addition, polynucleotides and polypeptides of the present inventionmay comprise one, two, three, four, five, six, seven, eight, or moremembrane domains.

Also, in preferred embodiments the present invention provides methodsfor further refining the biological function of the polynucleotidesand/or polypeptides of the present invention.

Specifically, the invention provides methods for using thepolynucleotides and polypeptides of the invention to identify orthologs,homologs, paralogs, variants, and/or allelic variants of the invention.Also provided are methods of using the polynucleotides and polypeptidesof the invention to identify the entire coding region of the invention,non-coding regions of the invention, regulatory sequences of theinvention, and secreted, mature, pro-, prepro-, forms of the invention(as applicable).

In preferred embodiments, the invention provides methods for identifyingthe glycosylation sites inherent in the polynucleotides and polypeptidesof the invention, and the subsequent alteration, deletion, and/oraddition of said sites for a number of desirable characteristics whichinclude, but are not limited to, augmentation of protein folding,inhibition of protein aggregation, regulation of intracellulartrafficking to organelles, increasing resistance to proteolysis,modulation of protein antigenicity, and mediation of intercellularadhesion.

In further preferred embodiments, methods are provided for evolving thepolynucleotides and polypeptides of the present invention usingmolecular evolution techniques in an effort to create and identify novelvariants with desired structural, functional, and/or physicalcharacteristics.

The present invention further provides for other experimental methodsand procedures currently available to derive functional assignments.These procedures include but are not limited to spotting of clones onarrays, micro-array technology, PCR based methods (e.g., quantitativePCR), anti-sense methodology, gene knockout experiments, and otherprocedures that could use sequence information from clones to build aprimer or a hybrid partner.

As used herein the terms “modulate or modulates” refer to an increase ordecrease in the amount, quality or effect of a particular activity, DNA,RNA, or protein.

Polynucleotides and Polypeptides of the Invention

Features of the Polypeptide Encoded by Gene No:1

Polypeptide fragments A and B corresponding to this gene provided as SEQID NO:2 and 4 (FIG. 1), encoded by the polynucleotide sequence accordingto SEQ ID NO:1 and 3 (FIG. 1), the predicted full-length polypeptidesequence corresponding to this gene provided as SEQ ID NO:150 (FIGS.20A-D), encoded by the full-length polynucleotide sequence according toSEQ ID NO:149 (FIGS. 20A-D), and/or encoded by the polynucleotidecontained within the deposited clone, BMY_HPP1, has significant homologyat the nucleotide and amino acid level to a number of phosphatases,which include, for example, the Schizosacchromyces Pombe proteintyrosine phosphatase PYP3 protein (PYP3_SP; Genbank Accession No:gi|P32587; SEQ ID NO:Y7); the mouse protein tyrosine phosphatase, receptortype, O, protein (MM_RPTPO; Genbank Accession No:gi| NP 035346; SEQ IDNO:Y8); and the human protein tyrosine phosphatase, receptor type, O,protein (HS_RPTPO; Genbank Accession No:gi| NP_(—)002839; SEQ ID NO:Y9);as determined by BLASTP. An alignment of the human phosphatasepolypeptide with these proteins is provided in FIGS. 6A-D. The conservedcatalytic residues are noted.

BMY_HPP1 is a novel phosphoprotein phosphatase encoded by a humangenomic BAC clone, Genbank accession AL360020. Aside from the predictedfull-length BMY_HPP1 polypeptide sequence, two separate homologousregions in BAC AL360020 have been identified. Fragment A of BMY_HPP1includes key conserved phosphatase catalytic residues: an Aspartate(“D”) at amino acid 11 of SEQ ID NO:2 (FIG. 1), a Cysteine (“C”) atamino acid 40 of SEQ ID NO:2 (FIG. 1), and an Arginine (“R”) at aminoacid 46 of SEQ ID NO:2 (FIG. 1). Fragment B of BMY_HPP1 represents amore N-terminal fragment and is not predicted to include any catalyticresidues. The predicted conserved phosphatase catalytic residues for thepredicted full-length BMY_HPP1 polypeptide are as follows: conservedphophatase catalytic residues: an Aspartate (“D”) at amino acid 14 ofSEQ ID NO:150 (FIGS. 20A-D), a Cysteine (“C”) at amino acid 42 of SEQ IDNO:150 (FIGS. 20A-D), and an Arginine (“R”) at amino acid 48 of SEQ IDNO:150 (FIGS. 20A-D).

An alignment of the BMY_HPP1 polypeptide fragments and predictedfull-length polypeptide with other phosphatase proteins (FIGS. 6A-D)illustrates the conserved phosphatase catalytic residues.

Based upon the strong homology to members of the phosphatase proteins,the polypeptide encoded by the human BMY_HPP1 phosphatase of the presentinvention is expected to share at least some biological activity withphosphatase proteins, preferably with members of the novelphosphotyrosine/dual-specificity (P-Tyr, P-Ser and P-Thr) phosphatases,particularly the novel phosphotyrosine/dual-specificity (P-Tyr, P-Serand P-Thr) phosphatases referenced herein.

The present invention encompasses the use of BMY_HPP1 inhibitors and/oractivators of BMY_HPP1 activity for the treatment, detection,amelioaration, or prevention of phosphatase associated disorders,including but not limited to metabolic diseases such as diabetes, inaddition to neural and/or cardiovascular diseases and disorders. Thepresent invention also encompasses the use of BMY_HPP1 inhibitors and/oractivators of BMY_HPP1 activity as immunosuppressive agents,anti-inflammatory agents, and/or anti-tumor agents

The present invention encompasses the use of BMY_HPP1 phosphataseinhibitors, including, antagonists such as antisense nucleic acids, inaddition to other antagonists, as described herein, in a therapeuticregimen to diagnose, prognose, treat, ameliorate, and/or preventdiseases where a kinase activity is insufficient. One, non-limitingexample of a disease which may occur due to insufficient kinase activityare certain types of diabetes, where one or more kinases involved in theinsulin receptor signal pathway may have insufficient activity orinsufficient expression, for example.

Moreover, the present invention encompasses the use of BMY_HPP1phosphatase activators, and/or the use of the BMY_HPP1 phosphatase geneor protein in a gene therapy regimen, as described herein, for thediagnoses, prognoses, treatment, amelioration, and/or prevention ofdiseases and/or disorders where a kinase activity is overly high, suchas a cancer where a kinase oncogene product has excessive activity orexcessive expression.

The present invention also encompasses the use of catalytically inactivevariants of BMY_HPP1 proteins, including fragments thereof, such as aprotein therapeutic, or the use of the encoding polynucleotide sequenceor as gene therapy, for example, in the diagnoses, prognosis, treatment,amelioration, and/or prevention of diseases or disorders wherephosphatase activity is overly high.

The present invention encompasses the use of antibodies directed againstthe BMY_HPP1 polypeptides, including fragment and/or variants thereof,of the present invention in diagnostics, as a biomarkers, and/or as atherapeutic agents.

The present invention encompasses the use of an inactive, non-catalytic,mutant of the BMY_HPP1 phosphatase as a substrate trapping mutant tobind cellular phosphoproteins or a library of phosphopeptides toidentify substrates of the BMY_HPP1 polypeptides.

The present invention encompasses the use of the BMY_HPP1 polypeptides,to identify inhibitors or activators of the BMY_HPP1 phosphataseactivity using either in vitro or ‘virtual’ (in silico) screeningmethods.

One embodiment of the invention relates to a method for identifying acompound as an activator or inhibitor of the BMY_HPP1 phosphatasecomprising the steps of: i.) contacting a BMY_HPP1 phosphatase inhibitoror activator labeled with an analytically detectable reagent with theBMY_HPP1 phosphatase under conditions sufficient to form a complex withthe inhibitor or activator; ii.) contacting said complex with a samplecontaining a compound to be identified; iii) and identifying thecompound as an inhibitor or activator by detecting the ability of thetest compound to alter the amount of labeled known BMY_HPP1 phosphataseinhibitor or activator in the complex.

Another embodiment of the invention relates to a method for identifyinga compound as an activator or inhibitor of a BMY_HPP1 phosphatasecomprising the steps of: i.) contacting the BMY_HPP1 phosphatase with acompound to be identified; and ii.) and measuring the ability of theBMY_HPP1 phosphatase to remove phosphate from a substrate.

The present invention also encomposses a method for identifying a ligandfor the BMY_HPP1 phosphatase comprising the steps of: i.) contacting theBMY_HPP1 phosphatase with a series of compounds under conditions topermit binding; and ii.) detecting the presence of any ligand-boundprotein.

Preferably, the above referenced methods comprise the BMY_HPP1phosphatase in a form selected from the group consisting of whole cells,cytosolic cell fractions, membrane cell fractions, purified or partiallypurified forms. The invention also relates to recombinantly expressedBMY_HPP1 phosphatase in a purified, substantially purified, orunpurified state. The invention further relates to BMY_HPP1 phosphatasefused or conjugated to a protein, peptide, or other molecule or compoundknown in the art, or referenced herein.

The present invention also encompasses pharmaceutical composition of theBMY_HPP1 phosphatase polypeptide comprising a compound identified byabove referenced methods and a pharmaceutically acceptable carrier.

Expression profiling designed to measure the steady state mRNA levelsencoding the BMY_HPP1 polypeptide showed predominately high expressionlevels in testis; to a significant extent, in the spinal cord, and to alesser extent, in pancreas, brain, pituitary, heart, and lung (as shownin FIG. 22).

Moreover, additional expression profiling of the BMY_HPP1 polypeptide innormal tissues showed strong expression in a number of brain subregionsand other central nervous system tissues, in particular the caudate,hippocampus and nucleus accumbens of the brain (as shown in FIG. 26).These regions are known to be involved in a number of neurologicaldisorders such as depression, bipolar disorder, schizophrenia, dementia,cognitive disorders and obesity. This data suggests a role formodulators of BMY_HPP1 activity in the treatment of neural disorders. Inaddition, BMY_HPP1 is strongly expressed in the adrenal, pineal andpituitary glands, suggesting a role for modulators of BMY_HPP1 activityin the treatment of endocrine disorders; in the atrium of the heart,suggesting a role for modulators of BMY_HPP1 activity in the treatmentof cardiac failure or other diseases of the heart; and in the testis,suggesting a role for modulators of BMY_HPP1 activity in the treatmentof male infertility caused by defective or insufficient spermatogenesis,as a contraceptive agent, or in the treatment of testicular cancer. Inaddition, BMY_HPP1 was expressed at lower levels across a number oftissues as well.

The strong homology to dual specificity phosphatases, combined with thepredominate localized expression in testis tissue emphasizes thepotential utility for BMY_HPP1 polynucleotides and polypeptides intreating, diagnosing, prognosing, and/or preventing testicular, inaddition to reproductive disorders.

In preferred embodiments, BMY_HPP1 polynucleotides and polypeptidesincluding agonists and fragments thereof, have uses which includetreating, diagnosing, prognosing, and/or preventing the following,non-limiting, diseases or disorders of the testis: spermatogenesis,infertility, Klinefelter's syndrome, XX male, epididymitis, genitalwarts, germinal cell aplasia, cryptorchidism, varicocele, immotile ciliasyndrome, and viral orchitis. The BMY_HPP1 polynucleotides andpolypeptides including agonists and fragments thereof, may also haveuses related to modulating testicular development, embryogenesis,reproduction, and in ameliorating, treating, and/or preventingtesticular proliferative disorders (e.g., cancers, which include, forexample, choriocarcinoma, Nonseminoma, seminona, and testicular germcell tumors).

Likewise, the predominate localized expression in testis tissue alsoemphasizes the potential utility for BMY_HPP1 polynucleotides andpolypeptides in treating, diagnosing, prognosing, and/or preventingmetabolic diseases and disorders which include the following, notlimiting examples: premature puberty, incomplete puberty, Kallmansyndrome, Cushing's syndrome, hyperprolactinemia, hemochromatosis,congenital adrenal hyperplasia, FSH deficiency, and granulomatousdisease, for example.

This gene product may also be useful in assays designed to identifybinding agents, as such agents (antagonists) are useful as malecontraceptive agents. The testes are also a site of active geneexpression of transcripts that is expressed, particularly at low levels,in other tissues of the body. Therefore, this gene product may beexpressed in other specific tissues or organs where it may play relatedfunctional roles in other processes, such as hematopoiesis,inflammation, bone formation, and kidney function, to name a fewpossible target indications.

The strong homology to dual specificity phosphatase proteins, combinedwith the localized expression in spinal cord, brain subregions, andother central nervous system tissues, suggests the BMY_HPP1polynucleotides and polypeptides may be useful in treating, diagnosing,prognosing, and/or preventing neurodegenerative disease states,behavioral disorders, or inflammatory conditions. Representative usesare described in the “Regeneration” and “Hyperproliferative Disorders”sections below, in the Examples, and elsewhere herein. Briefly, the usesinclude, but are not limited to the detection, treatment, and/orprevention of Alzheimer's Disease, Parkinson's Disease, Huntington'sDisease, Tourette Syndrome, meningitis, encephalitis, demyelinatingdiseases, peripheral neuropathies, neoplasia, trauma, congenitalmalformations, spinal cord injuries, ischemia and infarction, aneurysms,hemorrhages, schizophrenia, mania, dementia, paranoia, obsessivecompulsive disorder, depression, panic disorder, learning disabilities,ALS, psychoses, autism, and altered behaviors, including disorders infeeding, sleep patterns, balance, and perception. In addition, elevatedexpression of this gene product in regions of the brain indicates itplays a role in normal neural function. Potentially, this gene productis involved in synapse formation, neurotransmission, learning,cognition, homeostasis, or neuronal differentiation or survival.Furthermore, the protein may also be used to determine biologicalactivity, to raise antibodies, as tissue markers, to isolate cognateligands or receptors, to identify agents that modulate theirinteractions, in addition to its use as a nutritional supplement.Protein, as well as, antibodies directed against the protein may showutility as a tumor marker and/or immunotherapy targets for the abovelisted tissues.

The BMY_HPP1 polypeptide has been shown to comprise one glycosylationsites according to the Motif algorithm (Genetics Computer Group, Inc.).As discussed more specifically herein, protein glycosylation is thoughtto serve a variety of functions including: augmentation of proteinfolding, inhibition of protein aggregation, regulation of intracellulartrafficking to organelles, increasing resistance to proteolysis,modulation of protein antigenicity, and mediation of intercellularadhesion.

Asparagine glycosylation sites have the following consensus pattern,N-{P}-[ST]-{P}, wherein N represents the glycosylation site. However, itis well known that that potential N-glycosylation sites are specific tothe consensus sequence Asn-Xaa-Ser/Thr. However, the presence of theconsensus tripeptide is not sufficient to conclude that an asparagineresidue is glycosylated, due to the fact that the folding of the proteinplays an important role in the regulation of N-glycosylation. It hasbeen shown that the presence of proline between Asn and Ser/Thr willinhibit N-glycosylation; this has been confirmed by a recent statisticalanalysis of glycosylation sites, which also shows that about 50% of thesites that have a proline C-terminal to Ser/Thr are not glycosylated.Additional information relating to asparagine glycosylation may be foundin reference to the following publications, which are herebyincorporated by reference herein: Marshall R. D., Annu. Rev. Biochem.41:673-702 (1972); Pless D. D., Lennarz W. J., Proc. Natl. Acad. Sci.U.S.A. 74:134-138 (1977); Bause E., Biochem. J. 209:331-336 (1983);Gavel Y., von Heijne G., Protein Eng. 3:433-442 (1990); and Miletich J.P., Broze G. J. Jr., J. Biol. Chem. 265:11397-11404 (1990).

In preferred embodiments, the following asparagine glycosylation sitepolypeptide is encompassed by the present invention: LTPLRNISCCDPKA (SEQID NO:158). Polynucleotides encoding this polypeptide are also provided.The present invention also encompasses the use of this BMY_HPP1asparagine glycosylation site polypeptide as an immunogenic and/orantigenic epitope as described elsewhere herein.

The BMY_HPP1 polypeptides of the present invention were determined tocomprise several phosphorylation sites based upon the Motif algorithm(Genetics Computer Group, Inc.). The phosphorylation of such sites mayregulate some biological activity of the BMY_HPP1 polypeptide. Forexample, phosphorylation at specific sites may be involved in regulatingthe proteins ability to associate or bind to other molecules (e.g.,proteins, ligands, substrates, DNA, etc.). In the present case,phosphorylation may modulate the ability of the BMY_HPP1 polypeptide toassociate with other potassium channel alpha subunits, beta subunits, orits ability to modulate potassium channel function.

The BMY_HPP1 polypeptide was predicted to comprise four PKCphosphorylation sites using the Motif algorithm (Genetics ComputerGroup, Inc.). In vivo, protein kinase C exhibits a preference for thephosphorylation of serine or threonine residues. The PKC phosphorylationsites have the following consensus pattern: [ST]-x-[RK], where S or Trepresents the site of phosphorylation and ‘x’ an intervening amino acidresidue. Additional information regarding PKC phosphorylation sites canbe found in Woodget J. R., Gould K. L., Hunter T., Eur. J. Biochem.161:177-184 (1986), and Kishimoto A., Nishiyama K., Nakanishi H.,Uratsuji Y., Nomura H., Takeyama Y., Nishizuka Y., J. Biol. Chem.260:12492-12499 (1985); which are hereby incorporated by referenceherein.

In preferred embodiments, the following PKC phosphorylation sitepolypeptides are encompassed by the present invention: TLSFWSQKFGGLE(SEQ ID NO:159), VQNSRTPRSPLDC (SEQ ID NO:160), PLDCGSSKAQFLV (SEQ IDNO:161), and/or PTVYNTKKIFKHT (SEQ ID NO:162). Polynucleotides encodingthese polypeptides are also provided. The present invention alsoencompasses the use of these BMY_HPP1 PKC phosphorylation sitepolypeptides as immunogenic and/or antigenic epitopes as describedelsewhere herein.

In further confirmation of the human BMY_HPP1 polypeptide representing anovel human phosphatase polypeptide, the BMY_HPP1 polypeptide has beenshown to comprise a tyrosine specific protein phosphatase active sitedomain according to the Motif algorithm (Genetics Computer Group, Inc.).

Tyrosine specific protein phosphatases (EC 3.1.3.48) (PTPase) areenzymes that catalyze the removal of a phosphate group attached to atyrosine residue. These enzymes are very important in the control ofcell growth, proliferation, differentiation and transformation. Multipleforms of PTPase have been characterized and can be classified into twocategories: soluble PTPases and transmembrane receptor proteins thatcontain PTPase domain(s).

The currently known PTPases are listed below: Soluble PTPases, PTPN1(PTP-1B), PTPN2 (T-cell PTPase; TC-PTP), PTPN3 (H1) and PTPN4 (MEG),enzymes that contain an N-terminal band 4.1-like domain and could act atjunctions between the membrane and cytoskeleton, PTPN5 (STEP), PTPN6(PTP-1C; HCP; SHP) and PTPN11 (PTP-2C; SH-PTP3; Syp), enzymes whichcontain two copies of the SH2 domain at its N-terminal extremity (e.g.,the Drosophila protein corkscrew (gene csw) also belongs to thissubgroup), PTPN7 (LC-PTP; Hematopoietic protein-tyrosine phosphatase;HePTP), PTPN8 (70Z-PEP), PTPN9 (MEG2), PTPN12 (PTP-G1; PTP-P19), YeastPTP1, Yeast PTP2 which may be involved in the ubiquitin-mediated proteindegradation pathway, Fission yeast pyp1 and pyp2 which play a role ininhibiting the onset of mitosis, Fission yeast pyp3 which contributes tothe dephosphorylation of cdc2, Yeast CDC14 which may be involved inchromosome segregation, Yersinia virulence plasmid PTPAses (gene yopH),Autographa californica nuclear polyhedrosis virus 19 Kd PTPase, Dualspecificity PTPases, DUSP1 (PTPN10; MAP kinase phosphatase-1; MKP-1);which dephosphorylates MAP kinase on both Thr-183 and Tyr-185, DUSP2(PAC-1), a nuclear enzyme that dephosphorylates MAP kinases ERK1 andERK2 on both Thr and Tyr residues, DUSP3 (VHR), DUSP4 (HVH2), DUSP5(HVH3), DUSP6 (Pyst1; MKP-3), DUSP7 (Pyst2; MKP-X), Yeast MSG5, a PTPasethat dephosphorylates MAP kinase FUS3, Yeast YVH1, Vaccinia virus H1PTPase—a dual specificity phosphatase,

Structurally, all known receptor PTPases, are made up of a variablelength extracellular domain, followed by a transmembrane region and aC-terminal catalytic cytoplasmic domain. Some of the receptor PTPasescontain fibronectin type III (FN-III) repeats, immunoglobulin-likedomains, MAM domains or carbonic anhydrase-like domains in theirextracellular region. The cytoplasmic region generally contains twocopies of the PTPAse domain. The first seems to have enzymatic activity,while the second is inactive but seems to affect substrate specificityof the first. In these domains, the catalytic cysteine is generallyconserved but some other, presumably important, residues are not.

PTPase domains consist of about 300 amino acids. There are two conservedcysteines, the second one has been shown to be absolutely required foractivity. Furthermore, a number of conserved residues in its immediatevicinity have also been shown to be important.

A consensus sequence for tyrosine specific protein phophatases isprovided as follows:

[LIVMF]-H-C-x(2)-G-x(3)-[STC]-[STAGP]-x-[LIVMFY], wherein C is theactive site residue and “X” represents any amino acid.

Additional information related to tyrosine specific protein phosphatasedomains and proteins may be found in reference to the followingpublications Fischer E. H., Charbonneau H., Tonks N. K., Science253:401-406 (1991); Charbonneau H., Tonks N. K., Annu. Rev. Cell Biol.8:463-493 (1992); Trowbridge I. S., J. Biol. Chem. 266:23517-23520(1991); Tonks N. K., Charbonneau H., Trends Biochem. Sci. 14:497-500(1989); and Hunter T., Cell 58:1013-1016 (1989); which are herebyincorporated herein by reference in their entirety.

In preferred embodiments, the following tyrosine specific proteinphosphatase active site domain polypeptide is encompassed by the presentinvention: QEGKVIHCHAGLGRTGVLIAYLV (SEQ ID NO:163). Polynucleotidesencoding these polypeptides are also provided. The present inventionalso encompasses the use of this tyrosine specific protein phosphataseactive site domain polypeptide as an immunogenic and/or antigenicepitope as described elsewhere herein.

In preferred embodiments, the following N-terminal BMY_HPP1 deletionpolypeptides are encompassed by the present invention: M1-L607, E2-L607,A3-L607, G4-L607, I5-L607, Y6-L607, F7-L607, Y8-L607, N9-L607, F10-L607,G11-L607, W12-L607, K13-L607, D14-L607, Y15-L607, G16-L607, V17-L607,A18-L607, S19-L607, L20-L607, T21-L607, T22-L607, I23-L607, L24-L607,D25-L607, M26-L607, V27-L607, K28-L607, V29-L607, M30-L607, T31-L607,F32-L607, A33-L607, L34-L607, Q35-L607, E36-L607, G37-L607, K38-L607,V39-L607, A40-L607, I41-L607, H42-L607, C43-L607, H44-L607, A45-L607,G46-L607, L47-L607, G48-L607, R49-L607, T50-L607, G51-L607, V52-L607,L53-L607, I54-L607, A55-L607, C56-L607, Y57-L607, L58-L607, V59-L607,F60-L607, A61-L607, T62-L607, R63-L607, M64-L607, T65-L607, A66-L607,D67-L607, Q68-L607, A69-L607, I70-L607, I71-L607, F72-L607, V73-L607,R74-L607, A75-L607, K76-L607, R77-L607, P78-L607, N79-L607, S80-L607,I81-L607, Q82-L607, T83-L607, R84-L607, G85-L607, Q86-L607, L87-L607,L88-L607, C89-L607, V90-L607, R91-L607, E92-L607, F93-L607, T94-L607,Q95-L607, F96-L607, L97-L607, T98-L607, P99-L607, L100-L607, R101-L607,N102-L607, I103-L607, F104-L607, S105-L607, C106-L607, C107-L607,D108-L607, P109-L607, K110-L607, A111-L607, H112-L607, A113-L607,V114-L607, T115-L607, L116-L607, P117-L607, Q118-L607, Y119-L607,L120-L607, I121-L607, R122-L607, Q123-L607, R124-L607, H125-L607,L126-L607, L127-L607, H128-L607, G129-L607, Y130-L607, E131-L607,A132-L607, R133-L607, L134-L607, L135-L607, K136-L607, H137-L607,V138-L607, P139-L607, K140-L607, I141-L607, I142-L607, H143-L607,L144-L607, V145-L607, C146-L607, K147-L607, L148-L607, L149-L607,L150-L607, D151-L607, L152-L607, A153-L607, E154-L607, N155-L607,R156-L607, P157-L607, V158-L607, M159-L607, M160-L607, K161-L607,D162-L607, V163-L607, S164-L607, E165-L607, G166-L607, P167-L607,G168-L607, L169-L607, S170-L607, A171-L607, E172-L607, I173-L607,E174-L607, K175-L607, T176-L607, M177-L607, S178-L607, E179-L607,M180-L607, V181-L607, T182-L607, M183-L607, Q184-L607, L185-L607,D186-L607, K187-L607, E188-L607, L189-L607, L190-L607, R191-L607,H192-L607, D193-L607, S194-L607, D195-L607, V196-L607, S197-L607,N198-L607, P199-L607, P200-L607, N201-L607, P202-L607, T203-L607,A204-L607, V205-L607, A206-L607, A207-L607, D208-L607, F209-L607,D210-L607, N211-L607, R212-L607, G213-L607, M214-L607, I215-L607,F216-L607, S217-L607, N218-L607, E219-L607, Q220-L607, Q221-L607,F222-L607, D223-L607, P224-L607, L225-L607, W226-L607, K227-L607,R228-L607, R229-L607, N230-L607, V231-L607, E232-L607, C233-L607,L234-L607, Q235-L607, P236-L607, L237-L607, T238-L607, H239-L607,L240-L607, K241-L607, R242-L607, R243-L607, L244-L607, S245-L607,Y246-L607, S247-L607, D248-L607, S249-L607, D250-L607, L251-L607,K252-L607, R253-L607, A254-L607, E255-L607, N256-L607, L257-L607,L258-L607, E259-L607, Q260-L607, G261-L607, E262-L607, T263-L607,P264-L607, Q265-L607, T266-L607, V267-L607, P268-L607, A269-L607,Q270-L607, I271-L607, L272-L607, V273-L607, G274-L607, H275-L607,K276-L607, P277-L607, R278-L607, Q279-L607, Q280-L607, K281-L607,L282-L607, I283-L607, S284-L607, H285-L607, C286-L607, Y287-L607,I288-L607, P289-L607, Q290-L607, S291-L607, P292-L607, E293-L607,P294-L607, D295-L607, L296-L607, H297-L607, K298-L607, E299-L607,A300-L607, L301-L607, V302-L607, R303-L607, S304-L607, T305-L607,L306-L607, S307-L607, F308-L607, W309-L607, S310-L607, Q311-L607,S312-L607, K313-L607, F314-L607, G315-L607, G316-L607, L317-L607,E318-L607, G319-L607, L320-L607, K321-L607, D322-L607, N323-L607,G324-L607, S325-L607, P326-L607, I327-L607, F328-L607, H329-L607,G330-L607, R331-L607, I332-L607, I333-L607, P334-L607, K335-L607,E336-L607, A337-L607, Q338-L607, Q339-L607, S340-L607, G341-L607,A342-L607, F343-L607, S344-L607, A345-L607, D346-L607, V347-L607,S348-L607, G349-L607, S350-L607, H351-L607, S352-L607, P353-L607,G354-L607, E355-L607, P356-L607, V357-L607, S358-L607, P359-L607,S360-L607, F361-L607, A362-L607, N363-L607, V364-L607, H365-L607,K366-L607, D367-L607, P368-L607, N369-L607, P370-L607, A371-L607,H372-L607, Q373-L607, Q374-L607, V375-L607, S376-L607, H377-L607,C378-L607, Q379-L607, C380-L607, K381-L607, T382-L607, H383-L607,G384-L607, V385-L607, G386-L607, S387-L607, P388-L607, G389-L607,S390-L607, V391-L607, R392-L607, Q393-L607, N394-L607, S395-L607,R396-L607, T397-L607, P398-L607, R399-L607, S400-L607, P401-L607,L402-L607, D403-L607, C404-L607, G405-L607, S406-L607, S407-L607,P408-L607, K409-L607, A410-L607, Q411-L607, F412-L607, L413-L607,V414-L607, E415-L607, H416-L607, E417-L607, T418-L607, Q419-L607,D420-L607, S421-L607, K422-L607, D423-L607, L424-L607, S425-L607,E426-L607, A427-L607, A428-L607, S429-L607, H430-L607, S431-L607,A432-L607, L433-L607, Q434-L607, S435-L607, E436-L607, L437-L607,S438-L607, A439-L607, E440-L607, A441-L607, R442-L607, R443-L607,I444-L607, L445-L607, A446-L607, A447-L607, K448-L607, A449-L607,L450-L607, A451-L607, N452-L607, L453-L607, N454-L607, E455-L607,S456-L607, V457-L607, E458-L607, K459-L607, E460-L607, E461-L607,L462-L607, K463-L607, R464-L607, K465-L607, V466-L607, E467-L607,M468-L607, W469-L607, Q470-L607, K471-L607, E472-L607, L473-L607,N474-L607, S475-L607, R476-L607, D477-L607, G478-L607, A479-L607,W480-L607, E481-L607, R482-L607, I483-L607, C484-L607, G485-L607,E486-L607, R487-L607, D488-L607, P489-L607, F490-L607, I491-L607,L492-L607, C493-L607, S494-L607, L495-L607, M496-L607, W497-L607,S498-L607, W499-L607, V500-L607, E501-L607, Q502-L607, L503-L607,K504-L607, E505-L607, P506-L607, V507-I508-L607, T509-L607, K510-L607,E51-L607, D512-L607, V513-L607, D514-L607, M515-L607, L516-L607,V517-L607, D518-L607, R519-L607, R520-L607, A521-L607, D522-L607,A523-L607, A524-L607, E525-L607, A526-L607, L527-L607, F528-L607,L529-L607, L530-L607, E531-L607, K532-L607, G533-L607, Q534-L607,H535-L607, Q536-L607, T537-L607, I538-L607, L539-L607, C540-L607,V541-L607, L542-L607, H543-L607, C544-L607, I545-L607, V546-L607,N547-L607, L548-L607, Q549-L607, T550-L607, I551-L607, P552-L607,V553-L607, D554-L607, V555-L607, E556-L607, E557-L607, A558-L607,F559-L607, L560-L607, A561-L607, H562-L607, A563-L607, I564-L607, K565-L607, A566-L607, F567-L607, T568-L607, K569-L607, V570-L607, N571-L607,F572-L607, D573-L607, S574-L607, E575-L607, N576-L607, G577-L607,P578-L607, T579-L607, V580-L607, Y581-L607, N582-L607, T583-L607,L584-L607, K585-L607, K586-L607, I587-L607, F588-L607, K589-L607,H590-L607, T591-L607, L592-L607, E593-L607, E594-L607, K595-L607,R596-L607, K597-L607, M598-L607, T599-L607, K600-L607, and/or D601-L607of SEQ ID NO:150. Polynucleotide sequences encoding these polypeptidesare also provided. The present invention also encompasses the use ofthese N-terminal BMY_HPP1 deletion polypeptides as immunogenic and/orantigenic epitopes as described elsewhere herein.

In preferred embodiments, the following C-terminal BMY_HPP1 deletionpolypeptides are encompassed by the present invention: M1-L607, M1-G606,M1-P605, M1-K604, M1-P603, M1-G602, M1-D601, M1-K600, M1-T599, M1-M598,M1-K597, M1-R596, M1-K595, M1-E594, M1-E593, M1-L592, M1-T591, M1-H590,M1-K589, M1-F588, M1-I587, M1-K586, M1-K585, M1-L584, M1-T583, M1-N582,M1-Y581, M1-V580, M1-T579, M1-P578, M1-G577, M1-N576, M1-E575, M1-S574,M1-D573, M1-F572, M1-N571, M1-V570, M1-K569, M1-T568, M1-F567, M1-A566,M1-K565, M1-I564, M1-A563, M1-H562, M1-A561, M1-L560, M1-F559, M1-A558,M1-E557, M1-E556, M1-V555, M1-D554, M1-V553, M1-P552, M1-I551, M1-T550,M1-Q549, M1-L548, M1-N547, M1-V546, M1-I545, M1-C544, M1-H543, M1-L542,M1-V541, M1-C540, M1-L539, M1-I538, M1-T537, M1-Q536, M1-H535, M1-Q534,M1-G533, M1-K532, M1-E531, M1-L530, M1-L529, M1-F528, M1-L527, M1-A526,M1-E525, M1-A524, M1-A523, M1-D522, M1-A521, M1-R520, M1-R519, M1-D518,M1-V517, M1-L516, M1-M515, M1-D514, M1-V513, M1-D512, M1-E511, M1-K510,M1-T509, M1-I508, M1-V507, M1-P506, M1-E505, M1-K504, M1-L503, M1-Q502,M1-E501, M1-V500, M1-W499, M1-S498, M1-W497, M1-M496, M1-L495, M1-S494,M1-C493, M1-L492, M1-I491, M1-F490, M1-P489, M1-D488, M1- R487, M1-E486,M1-G485, M1-C484, M1-I483, M1-R482, M1-E481, M1-W480, M1-A479, M1-G478,M1-D477, M1-R476, M1-S475, M1-N474, M1-L473, M1-E472, M1-K471, M1-Q470,M1-W469, M1-M468, M1-E467, M1-V466, M1-K465, M1-R464, M1-K463, M1-L462,M1-E461, M1-E460, M1-K459, M1-E458, M1-V457, M1-S456, M1-E455, M1-N454,M1-L453, M1-N452, M1-A451, M1-L450, M1-A449, M1-K448, M1-A447, M1-A446,M1-L445, M1-I444, M1-R443, M1-R442, M1-A441, M1-E440, M1-A439, M1-S438,M1-L437, M1-E436, M1-S435, M1-Q434, M1-L433, M1-A432, M1-S431, M1-H430,M1-S429, M1-A428, M1-A427, M1-E426, M1-S425, M1-L424, M1-D423, M1-K422,M1-S421, M1-D420, M1-Q419, M1-T418, M1-E417, M1-H416, M1-E415, M1-V414,M1-L413, M1-F412, M1-Q411, M1-A410, M1-K409, M1-P408, M1-S407, M1-S406,M1-G405, M1-C404, M1-D403, M1-L402, M1-P401, M1-S400, M1-R399, M1-P398,M1-T397, M1-R396, M1-S395, M1-N394, M1-Q393, M1-R392, M1-V391, M1-S390,M1-G389, M1-P388, M1-S387, M1-G386, M1-V385, M1-G384, M1-H383, M1-T382,M1-K381, M1-C380, M1-Q379, M1-C378, M1-H377, M1-S376, M1-V375, M1-Q374,M1-Q373, M1-H372, M1-A371, M1-P370, M1-N369, M1-P368, M1-D367, M1-K366,M1-H365, M1-V364, M1-N363, M1-A362, M1-F361, M1-S360, M1-P359, M1-S358,M1-V357, M1-P356, M1-E355, M1-G354, M1-P353, M1-S352, M1-H351, M1-S350,M1-G349, M1-S348, M1-V347, M1-D346, M1-A345, M1-S344, M1-F343, M1-A342,M1-G341, M1-S340, M1-Q339, M1-Q338, M1-A337, M1-E336, M1-K335, M1-P334,M1-I333, M1-I332, M1-R331, M1-G330, M1-H329, M1-F328, M1-I327, M1-P326,M1-S325, M1-G324, M1-N323, M1-D322, M1-K321, M1-L320, M1-G319, M1-E318,M1-L317, M1-G316, M1-G315, M1-F314, M1-K313, M1-S312, M1-Q311, M1-S310,M1-W309, M1-F308, M1-S307, M1-L306, M1-T305, M1-S304, M1-R303, M1-V302,M1-L301, M1-A300, M1-E299, M1-K298, M1-H297, M1-L296, M1-D295, M1-P294,M1-E293, M1-P292, M1-S291, M1-Q290, M1-P289, M1-I288, M1-Y287, M1-C286,M1-H285, M1-S284, M1-I283, M1-L282, M1-K281, M1-Q280, M1-Q279, M1-R278,M1-P277, M1-K276, M1-H275, M1-G274, M1-V273, M1-L272, M1-I271, M1-Q270,M1-A269, M1-P268, M1-V267, M1-T266, M1-Q265, M1-P264, M1-T263, M1-E262,M1-G261, M1-Q260, M1-E259, M1-L258, M1-L257, M1-N256, M1-E255, M1-A254,M1-R253, M1-K252, M1-L251, M1-D250, M1-S249, M1-D248, M1-S247, M1-Y246,M1-S245, M1-L244, M1-R243, M1-R242, M1-K241, M1-L240, M1-H239, M1-T238,M1-L237, M1-P236, M1-Q235, M1-L234, M1-C233, M1-E232, M1-V231, M1-N230,M1-R229, M1-R228, M1-K227, M1-W226, M1-L225, M1-P224, M1-D223, M1-F222,M1-Q221, M1-Q220, M1-E219, M1-N218, M1-S217, M1-F216, M1-I215, M1-M214,M1-G213, M1-R212, M1-N211, M1-D210, M1-F209, M1-D208, M1-A207, M1-A206,M1-V205, M1-A204, M1-T203, M1-P202, M1-N201, M1-P200, M1-P199, M1-N198,M1-S197, M1-V196, M1-D195, M1-S194, M1-D193, M1-H192, M1-R191, M1-L190,M1-L189, M1-E188, M1-K187, M1-D186, M1-L185, M1-Q184, M1-M183, M1-T182,M1-V181, M1-M180, M1-E179, M1-S178, M1-M177, M1-T176, M1-K175, M1-E174,M1-I173, M1-E172, M1-A171, M1-S170, M1-L169, M1-G168, M1-P167, M1-G166,M1-E165, M1-S164, M1-V163, M1-D162, M1-K161, M1-M160, M1-M159, M1-V158,M1-P157, M1-R156, M1-N155, M1-E154, M1-A153, M1-L152, M1-D151, M1-L150,M1-L149, M1-L148, M1-K147, M1-C146, M1-V145, M1-L144, M1-H143, M1-I142,M1-I141, M1-K140, M1-P139, M1-V138, M1-H137, M1-K136, M1-L135, M1-L134,M1-R133, M1-A132, M1-E131, M1-Y130, M1-G129, M1-H128, M1-L127, M1-L126,M1-H125, M1-R124, M1-Q123, M1-R122, M1-I121, M1-L120, M1-Y119, M1-Q118,M1-P117, M1-L116, M1-T115, M1-V114, M1-A113, M1-H112, M1-A111, M1-K110,M1-P109, M1-D108, M1-C107, M1-C106, M1-S105, M1-F104, M1-I103, M1-N102,M1-R101, M1-L100, M1-P99, M1-T98, M1-L97, M1-F96, M1-Q95, M1-T94,M1-F93, M1-E92, M1-R91, M1-V90, M1-C89, M1-L88, M1-L87, M1-Q86, M1-G85,M1-R84, M1-T83, M1-Q82, M1-I81, M1-S80, M1-N79, M1-P78, M1-R77, M1-K76,M1-A75, M1-R74, M1-V73, M1-F72, M1-I71, M1-I70, M1-A69, M1-Q68, M1-D67,M1-A66, M1-T65, M1-M64, M1-R63, M1-T62, M1-A61, M1-F60, M1-V59, M1-L58,M1-Y57, M1-C56, M1-A55, M1-I54, M1-L53, M1-V52, M1-G51, M1-T50, M1-R49,M1-G48, M1-L47, M1-G46, M1-A45, M1-H44, M1-C43, M1-H42, M1-I41, M1-A40,M1-V39, M1-K38, M1-G37, M1-E36, M1-Q35, M1-L34, M1-A33, M1-F32, M1-T31,M1-M30, M1-V29, M1-K28, M1-V27, M1-M26, M1-D25, M1-L24, M1-I23, M1-T22,M1-T21, M1-L20, M1-S19, M1-A18, M1-V17, M1-G16, M1-Y15, M1-D14, M1-K13,M1-W12, M1-G11, M1-F10, M1-N9, M1-Y8, and/or M1-F7 of SEQ ID NO:150.Polynucleotide sequences encoding these polypeptides are also provided.The present invention also encompasses the use of these C-terminalBMY_HPP1 deletion polypeptides as immunogenic and/or antigenic epitopesas described elsewhere herein.

In preferred embodiments, the following polypeptide is encompassed bythe present invention:MEAGIYFNFGWKDYGVASLTTIDMVKVMTFALQEGKVIHCHAGLGRTGVLIAYLVFATRMTADQAIIVRAKRPNSIQTRGQLCVREFTQFLTPLRNISCCDPKAHAVTLPQYIRQRHLLHGYEARLLHVPKIIHLVCKLLLDAENRPVMMKDVSEGPLSAEIEKTMSEMVTMLDKELLRHDSDVSNPNPTAVAADFDNRGMISNEQQFDPLWKRRNVCLQPLTHLKRRLSYSSDLKRAENLLEQGETQTVPAQILVGHKPRQKLISHCYIPQSPEPDHKEALVRSTLSFWSQKFGGLEGLKDNGSPIHGRIIPKEAQQSGAFADVSGSHSPGEPVSPFANVHKDPNPAHQQVHCQCKTHGVGSPGSVQNSRTPRSPLDCGSSKAQFLVEHETQDSKDSEAASHSALQSELSAARRILAAKALANLNEVEKEELKRKVEMWQKLNSRDGAWERICGERP FILCSLMWSWVE(SEQ ID NO:153). Polynucleotides encoding these polypeptides are alsoprovided. The present invention also encompasses the use of thispolypeptide as an immunogenic and/or antigenic epitope as describedelsewhere herein.

In preferred embodiments, the following BMY_HPP1 phosphatase active sitedomain amino acid substitutions are encompassed by the presentinvention: wherein M1 is substituted with either an A, C, D, E, F, G, H,I, K, L, N, P, Q, R, S, T, V, W, or Y; wherein E2 is substituted witheither an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;wherein A3 is substituted with either a C, D, E, F, G, H, I, K, L, M, N,P, Q, R, S, T, V, W, or Y; wherein G4 is substituted with either an A,C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein I5 issubstituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S,T, V, W, or Y; wherein Y6 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein F7 is substitutedwith either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, orY; wherein N8 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, P, Q, R, S, T, V, W, or Y; wherein F9 is substituted with eitheran A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; whereinG10 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q,R, S, T, V, W, or Y; wherein W11 is substituted with either an A, C, D,E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein K12 issubstituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S,T, V, W, or Y; wherein D13 is substituted with either an A, C, E, F, G,H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Y14 is substitutedwith either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, orW; wherein G15 is substituted with either an A, C, D, E, F, H, I, K, L,M, N, P, Q, R, S, T, V, W, or Y; wherein V16 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; whereinA17 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q,R, S, T, V, W, or Y; wherein S18 is substituted with either an A, C, D,E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein L19 issubstituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S,T, V, W, or Y; wherein T20 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein T21 is substitutedwith either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, orY; wherein I22 is substituted with either an A, C, D, E, F, G, H, K, L,M, N, P, Q, R, S, T, V, W, or Y; wherein D23 is substituted with eitheran A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; whereinM24 is substituted with either an A, C, D, E, F, G, H, I, K, L, N, P, Q,R, S, T, V, W, or Y; wherein V25 is substituted with either an A, C, D,E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein K26 issubstituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S,T, V, W, or Y; wherein V27 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein M28 is substitutedwith either an A, C, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, orY; wherein T29 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, N, P, Q, R, S, V, W, or Y; wherein F30 is substituted with eitheran A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; whereinA31 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q,R, S, T, V, W, or Y; wherein L32 is substituted with either an A, C, D,E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein Q33 issubstituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S,T, V, W, or Y; wherein E34 is substituted with either an A, C, D, F, G,H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein G35 is substitutedwith either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, orY; wherein K36 is substituted with either an A, C, D, E, F, G, H, I, L,M, N, P, Q, R, S, T, V, W, or Y; wherein V37 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; whereinI38 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q,R, S, T, V, W, or Y; wherein H39 is substituted with either an A, C, D,E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein C40 issubstituted with either an A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,T, V, W, or Y; wherein H41 is substituted with either an A, C, D, E, F,G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A42 is substitutedwith either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, orY; wherein G43 is substituted with either an A, C, D, E, F, H, I, K, L,M, N, P, Q, R, S, T, V, W, or Y; wherein L44 is substituted with eitheran A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; whereinG45 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q,R, S, T, V, W, or Y; wherein R46 is substituted with either an A, C, D,E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein T47 issubstituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, V, W, or Y; wherein G48 is substituted with either an A, C, D, E, F,H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V49 is substitutedwith either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, orY; wherein L50 is substituted with either an A, C, D, E, F, G, H, I, K,M, N, P, Q, R, S, T, V, W, or Y; wherein I51 is substituted with eitheran A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; whereinA52 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q,R, S, T, V, W, or Y; wherein Y53 is substituted with either an A, C, D,E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein L54 issubstituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S,T, V, W, or Y; wherein V55 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein F56 is substitutedwith either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, orY; and/or wherein A57 is substituted with either a C, D, E, F, G, H, I,K, L, M, N, P, Q, R, S, T, V, W, or Y of SEQ ID NO:150, in addition toany combination thereof. The present invention also encompasses the useof these BMY_HPP1 phosphatase active site domain amino acid substitutedpolypeptides as immunogenic and/or antigenic epitopes as describedelsewhere herein.

In preferred embodiments, the following BMY_HPP1 phosphatase active sitedomain conservative amino acid substitutions are encompassed by thepresent invention: wherein M1 is substituted with either an A, G, S, orT; wherein E2 is substituted with a D; wherein A3 is substituted witheither a G, I, L, M, S, T, or V; wherein G4 is substituted with eitheran A, M, S, or T; wherein I5 is substituted with either an A, V, or L;wherein Y6 is either an F, or W; wherein F7 is substituted with either aW, or Y; wherein N8 is substituted with a Q; wherein F9 is substitutedwith either a W, or Y; wherein G10 is substituted with either an A, M,S, or T; wherein W11 is either an F, or Y; wherein K12 is substitutedwith either a R, or H; wherein D13 is substituted with an E; wherein Y14is either an F, or W; wherein G15 is substituted with either an A, M, S,or T; wherein V16 is substituted with either an A, I, or L; wherein A17is substituted with either a G, I, L, M, S, T, or V; wherein S18 issubstituted with either an A, G, M, or T; wherein L19 is substitutedwith either an A, I, or V; wherein T20 is substituted with either an A,G, M, or S; wherein T21 is substituted with either an A, G, M, or S;wherein I22 is substituted with either an A, V, or L; wherein D23 issubstituted with an E; wherein M24 is substituted with either an A, G,S, or T; wherein V25 is substituted with either an A, I, or L; whereinK26 is substituted with either a R, or H; wherein V27 is substitutedwith either an A, I, or L; wherein M28 is substituted with either an A,G, S, or T; wherein T29 is substituted with either an A, G, M, or S;wherein F30 is substituted with either a W, or Y; wherein A31 issubstituted with either a G, I, L, M, S, T, or V; wherein L32 issubstituted with either an A, I, or V; wherein Q33 is substituted with aN; wherein E34 is substituted with a D; wherein G35 is substituted witheither an A, M, S, or T; wherein K36 is substituted with either a R, orH; wherein V37 is substituted with either an A, I, or L; wherein 138 issubstituted with either an A, V, or L; wherein H39 is substituted witheither a K, or R; wherein C40 is a C; wherein H41 is substituted witheither a K, or R; wherein A42 is substituted with either a G, I, L, M,S, T, or V; wherein G43 is substituted with either an A, M, S, or T;wherein L44 is substituted with either an A, I, or V; wherein G45 issubstituted with either an A, M, S, or T; wherein R46 is substitutedwith either a K, or H; wherein T47 is substituted with either an A, G,M, or S; wherein G48 is substituted with either an A, M, S, or T;wherein V49 is substituted with either an A, I, or L; wherein L50 issubstituted with either an A, I, or V; wherein I51 is substituted witheither an A, V, or L; wherein A52 is substituted with either a G, I, L,M, S, T, or V; wherein Y53 is either an F, or W; wherein L54 issubstituted with either an A, I, or V; wherein V55 is substituted witheither an A, I, or L; wherein F56 is substituted with either a W, or Y;and/or wherein A57 is substituted with either a G, I, L, M, S, T, or Vof SEQ ID NO:150 in addition to any combination thereof. Other suitablesubstitutions within the BMY_HPP1 phosphatase active site domain areencompassed by the present invention and are referenced elsewhereherein. The present invention also encompasses the use of these BMY_HPP1phosphatase active site domain conservative amino acid substitutedpolypeptides as immunogenic and/or antigenic epitopes as describedelsewhere herein.

In addition, the present invention also encompasses BMY_HPP1polypeptides resulting from alternative initiating start codon positionsof the BMY_HPP1 polynucleotide (SEQ ID NO:149).

In preferred embodiments, the following polypeptide resulting from thestart codon beginning at nucleotide 31 of SEQ ID NO:149 is encompassedby the present invention:MQVQDATRRPSAVRFLSSFLQGRRHSTSDPVLRLQQARRGSGLGSGSATKLLSSSSLQVMVAVSSVSHAEGNPTFPERKRNLERPTPKYTKVGERLRHVIPGHMACSMACGGRACKYENPARWSEQEQAIKGVYSSWVTDNILAMARPSSELLEKYHIIDQFLSHGIKTIINLQRPGEHASCGNPLEQESGFTYLPEAFMEAGIYFYNFGWKDYGVASLTTILDMVKVMTFALQEGKVAIHCHAGLGRTGVLIACYLVFATRMTADQAIIFVRAKRPNSIQTRGQLLCVREFTQFLTPLRNIFSCCDPKAHAVTLPQYLIRQRHLLHGYEARLLKHVPKIIHLVCKLLLDLAENRPVMMKDVSEGPGLSAEIEKTMSEMVTMQLDKELLRHDSDVSNPPNPTAVAADFDNRGMIFSNEQQFDPLWKRRNVECLQPLTHLKRRLSYSDSDLKRAENLLEQGETPQTVPAQILVGHKPRQQKLISHCYIPQSPEPDLHKEALVRSTLSFWSQSKFGGLEGLKDNGSPIFHGRIIPKEAQQSGAFSADVSGSHSPGEPVSPSFANVHKDPNPAHQQVSHCQCKTHGVGSPGSVRQNSRTPRSPLDCGSSPKAQFLVEHETQDSKDLSEAASHSALQSELSAEARRILAAKALANLNESVEKEELKRKVEMWQKELNSRDGAWERICGERDPFILCSLMWSWVEQLKEPVITKEDVDMLVDRRADAAEALFLLEKGQHQTILCVLHClVNLQTIPVDVEEAFLAHAIKAFTKVNFDSENGPTVYNTLKKIFKHTLEEKRKMTKDGP KPGL (SEQ IDNO:175). Polynucleotides encoding these polypeptides are also provided.The present invention also encompasses the use of this polypeptide as animmunogenic and/or antigenic epitope as described elsewhere herein.

In preferred embodiments, the following polypeptide resulting from thestart codon beginning at nucleotide 208 of SEQ ID NO:149 is encompassedby the present invention:MVAVSSVSHAEGNPTFPERKRNLERPTPKYTKVGERLRHVIPGHMACSMACGGRACKYENPARWSEQEQAIKGVYSSWVTDNILAMARPSSELLEKYHIIDQFLSHGIKTIINLQRPGEHASCGNPLEQESGFTYLPEAFMEAGIYFYNFGWKDYGVASLTTILDMVKVMTFALQEGKVAIHCHAGLGRTGVLIACYLVFATRMTADQAIIFVRAKRPNSIQTRGQLLCVREFTQFLTPLRNIFSCCDPKAHAVTLPQYLIRQRHLLHGYEARLLKHVPKIIHLVCKLLLDLAENRPVMMKDVSEGPGLSAEIEKTMSEMVTMQLDKELLRHDSDVSNPPNPTAVAADFDNRGMIFSNEQQFDPLWKRRNVECLQPLTHLKRRLSYSDSDLKRAENLLEQGETPQTVPAQILVGHKPRQQKLISHCYIPQSPEPDLHKEALVRSTLSFWSQSKFGGLEGLKDNGSPIFHGRIIPKEAQQSGAFSADVSGSHSPGEPVSPSFANVHKDPNPAHQQVSHCQCKTHGVGSPGSVRQNSRTPRSPLDCGSSPKAQFLVEHETQDSKDLSEAASHSALQSELSAEARRILAAKALANLNESVEKEELKRKVEMWQKELNSRDGAWERICGERDPFILCSLMWSWVEQLKEPVITKEDVDMLVDRRADAAEALFLLEKGQHQTILCVLHCIVNLQTIPVDVEEAFLAHAIKAFTKVNFDSENGPTVYNTLKKIFKHTLEEKRKMTKDGPKP GL (SEQ IDNO:176). Polynucleotides encoding these polypeptides are also provided.The present invention also encompasses the use of this polypeptide as animmunogenic and/or antigenic epitope as described elsewhere herein.

In preferred embodiments, the following polypeptide resulting from thestart codon beginning at nucleotide 352 of SEQ ID NO:149 is encompassedby the present invention:MACGGRACKYENPARWSEQEQAIKGVYSSWVTDNILAMARPSSELLEKYHIIDQFLSHGIKTIINLQRPGEHASCGNPLEQESGFTYLPEAFMEAGIYFYNFGWKDYGVASLTTILDMVKVMTFALQEGKVAIHCHAGLGRTGVLIACYLVFATRMTADQAIIFVRAKRPNSIQTRGQLLCVREFTQFLTPLRNIFSCCDPKAHAVTLPQYLIRQRHLLHGYEARLLKHVPKIIHLVCKLLLDLAENRPVMMKDVSEGPGLSAEIEKTMSEMVTMQLDKELLRHDSDVSNPPNPTAVAADFDNRGMIFSNEQQFDPLWKRRNVECLQPLTHLKRRLSYSDSDLKRAENLLEQGETPQTVPAQILVGHKPRQQKLISHCYIPQSPEPDLHKEALVRSTLSFWSQSKFGGLEGLKDNGSPIFHGRIIPKEAQQSGAFSADVSGSHSPGEPVSPSFANVHKDPNPAHQQVSHCQCKTHGVGSPGSVRQNSRTPRSPLDCGSSPKAQFLVEHETQDSKDLSEAASHSALQSELSAEARRILAAKALANLNESVEKEELKRKVEMWQKELNSRDGAWERICGERDPFILCSLMWSWVEQLKEPVITKEDVDMLVDRRADAAEALFLLEKGQHQTILCVLHCIVNLQTIPVDVEEAFLAHAIKAFTKVNFDSENGPTVYNTLKKIFKHTLEEKRK MTKDGPKPGL(SEQ ID NO:177). Polynucleotides encoding these polypeptides are alsoprovided. The present invention also encompasses the use of thispolypeptide as an immunogenic and/or antigenic epitope as describedelsewhere herein.

In preferred embodiments, the following polypeptide resulting from thestart codon beginning at nucleotide 463 of SEQ ID NO:149 is encompassedby the present invention:MARPSSELLEKYHIIDQFLSHGIKTIINLQRPGEHASCGNPLEQESGFTYLPEAFMEAGIYFYNFGWKDYGVASLTTILDMVKVMTFALQEGKVAIHCHAGLGRTGVLIACYLVFATRMTADQAIIFVRAKRPNSIQTRGQLLCVREFTQFLTPLRNIFSCCDPKAHAVTLPQYLIRQRHLLHGYEARLLKHVPKIIHLVCKLLLDLAENRPVMMKDVSEGPGLSAEIEKTMSEMVTMQLDKELLRHDSDVSNPPNPTAVAADFDNRGMIFSNEQQFDPLWKRRNVECLQPLTHLKRRLSYSDSDLKRAENLLEQGETPQTVPAQILVGHKPRQQKLISHCYIPQSPEPDLHKEALVRSTLSFWSQSKFGGLEGLKDNGSPIFHGRIIPKEAQQSGAFSADVSGSHSPGEPVSPSFANVHKDPNPAHQQVSHCQCKTHGVGSPGSVRQNSRTPRSPLDCGSSPKAQFLVEHETQDSKDLSEAASHSALQSELSAEARRILAAKALANLNESVEKEELKRKVEMWQKELNSRDGAWERICGERDPFILCSLMWSWVEQLKEPVITKEDVDMLVDRRADAAEALFLLEKGQHQTILCVLHClVNLQTIPVDVEEAFLAHAIKAFTKVNFDSENGPTVYNTLKKIFKHTLEEKRKMTKDGPKPGL (SEQ ID NO:178). Polynucleotides encodingthese polypeptides are also provided. The present invention alsoencompasses the use of this polypeptide as an immunogenic and/orantigenic epitope as described elsewhere herein.

In preferred embodiments, the present invention encompasses apolynucleotide lacking the initiating start codon, in addition to, theresulting encoded polypeptide of BMY_HPP1. Specifically, the presentinvention encompasses the polynucleotide corresponding to nucleotides631 thru 2448 of SEQ ID NO:149, and the polypeptide corresponding toamino acids 2 thru 607 of SEQ ID NO:150. Also encompassed arerecombinant vectors comprising said encoding sequence, and host cellscomprising said vector.

The present invention also provides a three-dimensional homology modelof the BMY_HPP1 polypeptide (see FIG. 28) representing amino acids M1 toE301 of BMY_HPP1 (SEQ ID NO:150). A three-dimensional homology model canbe constructed on the basis of the known structure of a homologousprotein (Greer et al, 1991, Lesk, et al, 1992, Cardozo, et al, 1995,Yuan, et al, 1995). The homology model of the BMY_HPP1 polypeptide,corresponding to amino acid residues M1 to E301 of SEQ ID NO:150, wasbased upon the homologous structure of 1aax, a Human Protein TyrosinePhosphatase Complex (residues D11-N321; Protein Data Bank, PDB entry1aax chain A; Genbank Accession No. gi|2981942; SEQ ID NO:206) and isdefined by the set of structural coordinates set forth in Table VIIIherein.

Homology models are useful when there is no experimental informationavailable on the protein of interest. A 3-dimensional model can beconstructed on the basis of the known structure of a homologous protein(Greer et. al., 1991, Lesk, et. al., 1992, Cardozo, et. al., 1995, Sali,et. al., 1995).

Those of skill in the art will understand that a homology model isconstructed on the basis of first identifying a template, or, protein ofknown structure which is similar to the protein without known structure.This can be accomplished through pairwise alignment of sequences usingsuch programs as FASTA (Pearson, et. al. 1990) and BLAST (Altschul, et.al., 1990). In cases where sequence similarity is high (greater than30%), these pairwise comparison methods may be adequate. Likewise,multiple sequence alignments or profile-based methods can be used toalign a query sequence to an alignment of multiple (structurally andbiochemically) related proteins. When the sequence similarity is low,more advanced techniques are used such as fold recognition (proteinthreading; Hendlich, et. al., 1990), where the compatibility of aparticular sequence with the 3-dimensional fold of a potential templateprotein is gauged on the basis of a knowledge-based potential. Followingthe initial sequence alignment, the query template can be optimallyaligned by manual manipulation or by incorporation of other features(motifs, secondary structure predictions, and allowed sequenceconservation). Next, structurally conserved regions can be identifiedand are used to construct the core secondary structure (Sali, et. al.,1995) elements in the three dimensional model. Variable regions, called“unconserved regions” and loops can be added using knowledge-basedtechniques. The complete model with variable regions and loops can berefined performing forcefield calculations (Sali, et. al., 1995,Cardozo, et. al., 1995).

Protein threading and molecular modeling of BMY_HPP1 suggested that aportion of BMY_HPP1 (residues M1 to E301) had a three dimensional foldsimilar to that of 1aax, a Human Protein Tyrosine Phosphatase Complex(residues D11-N321; Protein Data Bank, PDB entry 1aax chain A; GenbankAccession No. gi|2981942; SEQ ID NO:206). Based on sequence, structureand known phosphatase signature sequences, BMY_HPP1 is a novel tyrosinespecific phosphatase.

For BMY_HPP1, the pairwise alignment method FASTA (Pearson, et. al.1990) and fold recognition methods (protein threading) generatedidentical sequence alignments for a portion (residues M1 to E301) ofBMY_HPP1 aligned with the sequence of 1aax a tyrosine specificphosphatase (residues D11-N321; Protein Data Bank, PDB entry 1aax chainA; Genbank Accession No. gi|2981942; SEQ ID NO:206). The alignment ofBMY_HPP1 with PDB entry 1aax is set forth in FIG. 27. In this invention,the homology model of BMY_HPP1 was derived from the sequence alignmentset forth in FIG. 27 (residues D11-N321 of SEQ ID NO:206). An overallatomic model including plausible sidechain orientations was generatedusing the program LOOK (Levitt 1992). The three dimensional model forBMY_HPP1 is defined by the set of structure coordinates as set forth inTable VIII and is shown in FIG. 28 rendered by backbone secondarystructures.

In order to recognize errors in three-dimensional structure, knowledgebased mean fields can be used to judge the quality of protein folds(Sippl 1993). The methods can be used to recognize misfolded structuresas well as faulty parts of structural models. The technique generates anenergy graph where the energy distribution for a given protein fold isdisplayed on the y-axis and residue position in the protein fold isdisplayed on the x-axis. The knowledge based mean fields compose a forcefield derived from a set of globular protein structures taken as asubset from the Protein Data Bank (Bernstein et. al. 1977). To analyzethe quality of a model the energy distribution is plotted and comparedto the energy distribution of the template from which the model wasgenerated. FIG. 29 shows the energy graph for the BMY_HPP1 model (dottedline) and the template (1aax, a tyrosine specific phosphatase) fromwhich the model was generated. It is clear that the model has slightlyhigher energies in the C-terminal region while the N-terminal regionappears to be “native-like”. This graph supports the motif and sequencealignments in confirming that the three dimensional structurecoordinates of BMY_HPP1 are an accurate and useful representation forthe polypeptide.

The term “structure coordinates” refers to Cartesian coordinatesgenerated from the building of a homology model.

Those of skill in the art will understand that a set of structurecoordinates for a protein is a relative set of points that define ashape in three dimensions. Thus, it is possible that an entirelydifferent set of coordinates could define a similar or identical shape.Moreover, slight variations in the individual coordinates, as emanatefrom generation of similar homology models using different alignmenttemplates (i.e., other than the structure coordinates of 1aax), and/orusing different methods in generating the homology model, will haveminor effects on the overall shape. Variations in coordinates may alsobe generated because of mathematical manipulations of the structurecoordinates. For example, the structure coordinates set forth in TableVIII and shown in FIG. 28 could be manipulated by fractionalization ofthe structure coordinates; integer additions or subtractions to sets ofthe structure coordinates, inversion of the structure coordinates or anycombination of the above.

Various computational analyses are therefore necessary to determinewhether a molecule or a portion thereof is sufficiently similar to allor parts of BMY_HPP1 described above as to be considered the same. Suchanalyses may be carried out in current software applications, such asINSIGHTII (Accelrys Inc., San Diego, Calif.) version 2000 or softwareapplications available in the SYBYL software suite (Tripos Inc., St.Louis, Mo.).

Using the superimposition tool in the program INSIGHTII comparisons canbe made between different structures and different conformations of thesame structure. The procedure used in INSIGHTII to compare structures isdivided into four steps: 1) load the structures to be compared; 2)define the atom equivalencies in these structures; 3) perform a fittingoperation; and 4) analyze the results. Each structure is identified by aname. One structure is identified as the target (i.e., the fixedstructure); the second structure (i.e., moving structure) is identifiedas the source structure. Since atom equivalency within INSIGHTII isdefined by user input, for the purpose of this invention we will defineequivalent atoms as protein backbone atoms (N, Cα, C and O) for allconserved residues between the two structures being compared. We willalso consider only rigid fitting operations. When a rigid fitting methodis used, the working structure is translated and rotated to obtain anoptimum fit with the target structure. The fitting operation uses analgorithm that computes the optimum translation and rotation to beapplied to the moving structure, such that the root mean squaredifference of the fit over the specified pairs of equivalent atom is anabsolute minimum. This number, given in angstroms, is reported byINSIGHTII. For the purpose of this invention, any homology model of aBMY_HPP1 that has a root mean square deviation of conserved residuebackbone atoms (N, Cα, C, O) of less than 3.0 A when superimposed on therelevant backbone atoms described by structure coordinates listed inTable VIII and shown in FIG. 28 are considered identical. Morepreferably, the root mean square deviation is less than 2.0 Å.

This invention as embodied by the homology model enables thestructure-based design of modulators of the biological function ofBMY_HPP1, as well as mutants with altered biological function and/orspecificity.

There is 18% sequence identity between catalytic domain of BMY_HPP1 andthe Human Protein Tyrosine Phosphatase 1B (PTP1B; PDB code 1aax) asdetermined using the GAP program within GCG (Genetics Computing Group,Wisconsin). The structure was used as the template to generate the threedimensional model for BMY_HPP1. For BMY_HPP1, the functionally importantresidues are located in a cleft near the site that in other phosphatasesis shown to be the active site. The active site residues are defined by:H189-C190-G193-R196 and D 161 as well as Y162. All these residues areconserved in PTP1B (denoted by the “*” in FIG. 27) and other knownphosphatases. In the 1aax polypeptide, the Cysteine was mutated to aSerine to facilitate structural analysis (Jia, Z., et al., 1995). Theseactive site residues play critical roles in the mechanism of catalysisand substrate specificity and binding.

In a preferred embodiment of the present invention, the moleculecomprises the active site region defined by structure coordinates ofBMY_HPP1 amino acids described above according to Table VIII, or amutant of said molecule. The active site is defined by residuesH189-C190-G193-R196 and D 161 as well as Y162 of SEQ ID NO:150. Based onthe sequence alignment disclosed in FIG. 27 and the structural modeldisclosed in Table VIII and visualized in FIG. 28, D161 is identified asa general acid, Y162 as a key determinant of substrate specificity whichinteracts with the phosphotyrosine substrate, C190 as the catalyticCysteine nucleophile which cleaves the phosphodiester bond, and R196 asthe essential Argenine which activates the bond for cleavage asdescribed in the literature (reviewed by Fauman and Saper, 1996).

More preferred are molecules comprising all or any part of the activesite region or a mutant or homologue of said molecule or molecularcomplex. By mutant or homologue of the molecule it is meant a moleculethat has a root mean square deviation from the backbone atoms of saidBMY_HPP1 amino acids of not more than 3.5 Angstroms.

More preferred are molecules comprising all or any part of the activesite region defined as residues above or a mutant or homologue of saidmolecule or molecular complex. By mutant or homologue of the molecule itis meant a molecule that has a root mean square deviation from thebackbone atoms of said residues in the active site region of saidBMY_HPP1 of not more than 3.5 Angstroms.

The term “root mean square deviation” means the square root of thearithmetic mean of the squares of the deviations from the mean. It is away to express the deviation or variation from a trend or object. Forpurposes of this invention, the “root mean square deviation” defines thevariation in the backbone of a protein from the relevant portion of thebackbone of BMY_HPP1 as defined by the structure coordinates describedherein.

The structure coordinates of a BMY_HPP1 homology model portion thereofare stored in a machine-readable storage medium. Such data may be usedfor a variety of purposes, such as drug discovery and targetprioritization and validation.

Accordingly, in one embodiment of this invention is provided amachine-readable data storage medium comprising a data storage materialencoded with the structure coordinates set forth in Table VIII andvisualized in FIG. 28.

One embodiment utilizes System 10 as disclosed in WO 98/11134, thedisclosure of which is incorporated herein by reference in its entirety.Briefly, one version of these embodiments comprises a computercomprising a central processing unit (“CPU”), a working memory which maybe, e.g, RAM (random-access memory) or “core” memory, mass storagememory (such as one or more disk drives or CD-ROM drives), one or morecathode-ray tube (“CRT”) display terminals, one or more keyboards, oneor more input lines, and one or more output lines, all of which areinterconnected by a conventional bidirectional system bus.

Input hardware, coupled to the computer by input lines, may beimplemented in a variety of ways. Machine-readable data of thisinvention may be inputted via the use of a modem or modems connected bya telephone line or dedicated data line. Alternatively or additionally,the input hardware may comprise CD-ROM drives or disk drives. Inconjunction with a display terminal, keyboard may also be used as aninput device.

Output hardware, coupled to the computer by output lines, may similarlybe implemented by conventional devices. By way of example, outputhardware may include a CRT display terminal for displaying a graphicalrepresentation of a region or domain of the present invention using aprogram such as QUANTA as described herein. Output hardware might alsoinclude a printer, so that hard copy output may be produced, or a diskdrive, to store system output for later use.

In operation, the CPU coordinates the use of the various input andoutput devices, coordinates data accesses from mass storage, andaccesses to and from the working memory, and determines the sequence ofdata processing steps. A number of programs may be used to process themachine-readable data of this invention. Such programs are discussed inreference to the computational methods of drug discovery as describedherein. Specific references to components of the hardware system areincluded as appropriate throughout the following description of the datastorage medium.

For the purpose of the present invention, any magnetic data storagemedium which can be encoded with machine-readable data would besufficient for carrying out the storage requirements of the system. Themedium could be a conventional floppy diskette or hard disk, having asuitable substrate, which may be conventional, and a suitable coating,which may be conventional, on one or both sides, containing magneticdomains whose polarity or orientation could be altered magnetically, forexample. The medium may also have an opening for receiving the spindleof a disk drive or other data storage device.

The magnetic domains of the coating of a medium may be polarized ororiented so as to encode in a manner which may be conventional, machinereadable data such as that described herein, for execution by a systemsuch as the system described herein.

Another example of a suitable storage medium which could also be encodedwith such machine-readable data, or set of instructions, which could becarried out by a system such as the system described herein, could be anoptically-readable data storage medium. The medium could be aconventional compact disk read only memory (CD-ROM) or a rewritablemedium such as a magneto-optical disk which is optically readable andmagneto-optically writable. The medium preferably has a suitablesubstrate, which may be conventional, and a suitable coating, which maybe conventional, usually of one side of substrate.

In the case of a CD-ROM, as is well known, the coating is reflective andis impressed with a plurality of pits to encode the machine-readabledata. The arrangement of pits is read by reflecting laser light off thesurface of the coating. A protective coating, which preferably issubstantially transparent, is provided on top of the reflective coating.

In the case of a magneto-optical disk, as is well known, the coating hasno pits, but has a plurality of magnetic domains whose polarity ororientation can be changed magnetically when heated above a certaintemperature, as by a laser. The orientation of the domains can be readby measuring the polarization of laser light reflected from the coating.The arrangement of the domains encodes the data as described above.

Thus, in accordance with the present invention, data capable ofdisplaying the three dimensional structure of the BMY_HPP1 homologymodel, or portions thereof and their structurally similar homologues isstored in a machine-readable storage medium, which is capable ofdisplaying a graphical three-dimensional representation of thestructure. Such data may be used for a variety of purposes, such as drugdiscovery.

For the first time, the present invention permits the use, throughhomology modeling based upon the sequence of BMY_HPP1 (FIGS. 20A-D) ofstructure-based or rational drug design techniques to design, select,and synthesizes chemical entities that are capable of modulating thebiological function of BMY_HPP1. Comparison of the BMY_HPP1 homologymodel with the structures of template phosphatases enable the use ofrational or structure based drug design methods to design, select orsynthesize specific chemical modulators of BMY_HPP1.

Accordingly, the present invention is also directed to the entiresequence in FIG. 20A-D or any portion thereof for the purpose ofgenerating a homology model for the purpose of three dimensionalstructure-based drug designs.

For purposes of this invention, we include mutants or homologues of thesequence in FIGS. 20A-D or any portion thereof. In a preferredembodiment, the mutants or homologues have at least 25% identity, morepreferably 50% identity, more preferably 75% identity, and mostpreferably 90% identity to the amino acid residues in FIGS. 20A-D (SEQID NO:150).

The three-dimensional model structure of the BMY_HPP1 will also providemethods for identifying modulators of biological function. Variousmethods or combination thereof can be used to identify these compounds.

Structure coordinates of the active site region defined above can alsobe used to identify structural and chemical features. Identifiedstructural or chemical features can then be employed to design or selectcompounds as potential BMY_HPP1 modulators. By structural and chemicalfeatures it is meant to include, but is not limited to, van der Waalsinteractions, hydrogen bonding interactions, charge interaction,hydrophobic interactions, and dipole interaction. Alternatively, or inconjunction, the three-dimensional structural model can be employed todesign or select compounds as potential BMY_HPP1 modulators. Compoundsidentified as potential BMY_HPP1 modulators can then be synthesized andscreened in an assay characterized by binding of a test compound to theBMY_HPP1, or in characterizing BMY_HPP1 deactivation in the presence ofa small molecule. Examples of assays useful in screening of potentialBMY_HPP1 modulators include, but are not limited to, screening insilico, in vitro assays and high throughput assays. Finally, thesemethods may also involve modifying or replacing one or more amino acidsfrom BMY_HPP1 according to Table VIII.

However, as will be understood by those of skill in the art upon thisdisclosure, other structure based design methods can be used. Variouscomputational structure based design methods have been disclosed in theart.

For example, a number of computer modeling systems are available inwhich the sequence of the BMY_HPP1 and the BMY_HPP1 structure (i.e.,atomic coordinates of BMY_HPP1 and/or the atomic coordinates of theactive site region as provided in Table VIII) can be input. The computersystem then generates the structural details of one or more theseregions in which a potential BMY_HPP1 modulator binds so thatcomplementary structural details of the potential modulators can bedetermined. Design in these modeling systems is generally based upon thecompound being capable of physically and structurally associating withBMY_HPP1. In addition, the compound must be able to assume aconformation that allows it to associate with BMY_HPP1. Some modelingsystems estimate the potential inhibitory or binding effect of apotential BMY_HPP1 modulator prior to actual synthesis and testing.

Methods for screening chemical entities or fragments for their abilityto associate with a given protein target are well known. Often thesemethods begin by visual inspection of the binding site on the computerscreen. Selected fragments or chemical entities are then positioned inone or more positions and orientations within the active site region inBMY_HPP1. Molecular docking is accomplished using software such asINSIGHTII, ICM (Molsoft LLC, La Jolla, Calif.), and SYBYL, following byenergy minimization and molecular dynamics with standard molecularmechanic forcefields such as CHARMM and MMFF. Examples of computerprograms which assist in the selection of chemical fragment or chemicalentities useful in the present invention include, but are not limitedto, GRID (Goodford, 1985), AUTODOCK (Goodsell, 1990), and DOCK (Kuntzet. al. 1982).

Alternatively, compounds may be designed de novo using either an emptyactive site or optionally including some portion of a known inhibitor.Methods of this type of design include, but are not limited to LUDI(Bohm 1992), LeapFrog (Tripos Associates, St. Louis Mo.) and DOCK (Kuntzet. al., 1982). Programs such as DOCK (Kuntz et. al. 1982) can be usedwith the atomic coordinates from the homology model to identifypotential ligands from databases or virtual databases which potentiallybind the in the active site region, and which may therefore be suitablecandidates for synthesis and testing. The computer programs may utilizea combination of the following steps:

(a) Selection of fragments or chemical entities from a database and thenpositioning the chemical entity in one or more orientations within theBMY_HPP1 active site defined by residues D161-Y162 andH189-C190-G193-R196. Characterization of the structural and chemicalfeatures of the chemical entity and active site including van der Waalsinteractions, hydrogen bonding interactions, charge interaction,hydrophobic bonding interaction, and dipole interactions;

(b) Search databases for molecular fragments which can be joined to orreplace the docked chemical entity and spatially fit into regionsdefined by the said BMY_HPP1 active site;

(c) Evaluate the docked chemical entity and fragments using acombination of scoring schemes which account for van der Waalsinteractions, hydrogen bonding interactions, charge interaction,hydrophobic interactions; or

(d) Databases that may be used include ACD (Molecular Designs Limited),Aldrich (Aldrich Chemical Company), NCI (National Cancer Institute),Maybridge (Maybridge Chemical Company Ltd), CCDC (CambridgeCrystallographic Data Center), CAST (Chemical Abstract Service), andDerwent (Derwent Information Limited).

Upon selection of preferred chemical entities or fragments, theirrelationship to each other and BMY_HPP1 can be visualized and thenassembled into a single potential modulator. Programs useful inassembling the individual chemical entities include, but are not limitedto SYBYL and LeapFrog (Tripos Associates, St. Louis Mo.), LUDI (Bohm1992) as well as 3D Database systems (Martin 1992).

Additionally, the three-dimensional homology model of BMY_HPP1 will aidin the design of mutants with altered biological activity. Site directedmutagenesis can be used to generate proteins with similar or varyingdegrees of biological activity compared to native BMY_HPP1. Thisinvention also relates to the generation of mutants or homologues ofBMY_HPP1. It is clear that molecular modeling using the threedimensional structure coordinates set forth in Table VIII andvisualization of the BMY_HPP1 model, FIG. 28 can be utilized to designhomologues or mutant polypeptides of BMY_HPP1 that have similar oraltered biological activities, function or reactivities.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:149 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides consisting of a nucleotide sequence described bythe general formula of a−b, where a is any integer between 1 to 4379 ofSEQ ID NO:149, b is an integer between 15 to 4393, where both a and bcorrespond to the positions of nucleotide residues shown in SEQ IDNO:149, and where b is greater than or equal to a+14.

Features of the Polypeptide Encoded by Gene No:2

The polypeptide fragment corresponding to this gene provided as SEQ IDNO:6 (FIG. 2), encoded by the polynucleotide sequence according to SEQID NO:5 (FIG. 2), and/or encoded by the polynucleotide contained withinthe deposited clone, BMY_HPP2, has significant homology at thenucleotide and amino acid level to a number of phosphatases, whichinclude, for example, the human CDC14 (also known as the cell divisioncycle 14, S. cerevisiae Gene A protein) homologue A (HS_CDC14A; GenbankAccession No:gi| NP_(—)003663; SEQ ID NO:30); the human S. cerevisiaeCDC14 homolog, gene B (HS_CDC14B; Genbank Accession No:gi| NP_(—)003662;SEQ ID NO:31); and the yeast soluble tyrosine-specific proteinphosphatase Cdc14p protein (SC_CDC14; Genbank Accession No:gi|NP_(—)002839; SEQ ID NO:32) as determined by BLASTP An alignment of thehuman phosphatase polypeptide with these proteins is provided in FIG. 7.

BMY_HPP2 is predicted to be a phosphoprotein phosphatase based on itshomology to human CDC14B as determined by BLASTP. BMY_HPP2 showssignificant homology to the catalytic domains of human CDC14A and CDC14Band to yeast CDC14 including a conserved Aspartate at AA 76, a Cysteineat AA106 and an Arginine at AA 112 of BMY_HPP2 (shown in FIG. 2).

Polypeptide sequences corresponding to portions of the encoded BMY_HPP2polypeptide sequence have been described as BAA91172 (Genbank AccessionNo:gi 7020545). However, conceptual translation of BAA91172 indicatesthat the phosphatase homology is in an open reading frame that beginsbefore the 5′ end of the provided polynucleotide EST sequence, inaddition to regions of the polypeptide that are homologous to knownphosphatases. Thus, the Genbank record, or the sequence, provided forBAA91172 does not provide any suggestion that this clone partiallyencodes a phosphatase protein.

Based upon the strong homology to members of the phosphatase proteins,the polypeptide encoded by the human BMY_HPP2 phosphatase of the presentinvention is expected to share at least some biological activity withphosphatase proteins, preferably with members of the novelphosphotyrosine/dual-specificity (P-Tyr, P-Ser and P-Thr) phosphatases,particularly the novel phosphotyrosine/dual-specificity (P-Tyr, P-Serand P-Thr) phosphatases referenced herein.

The present invention encompasses the use of BMY_HPP2 inhibitors and/oractivators of BMY_HPP2 activity for the treatment, detection,amelioaration, or prevention of phosphatase associated disorders,including but not limited to metabolic diseases such as diabetes, inaddition to neural and/or cardiovascular diseases and disorders. Thepresent invention also encompasses the use of BMY_HPP2 inhibitors and/oractivators of BMY_HPP2 activity as immunosuppressive agents,anti-inflammatory agents, and/or anti-tumor agents.

The present invention encompasses the use of BMY_HPP2 phosphataseinhibitors, including, antagonists such as antisense nucleic acids, inaddition to other antagonists, as described herein, in a therapeuticregimen to diagnose, prognose, treat, ameliorate, and/or preventdiseases where a kinase activity is insufficient. One, non-limitingexample of a disease which may occur due to insufficient kinase activityare certain types of diabetes, where one or more kinases involved in theinsulin receptor signal pathway may have insufficient activity orinsufficient expression, for example.

Moreover, the present invention encompasses the use of BMY_HPP2phosphatase activators, and/or the use of the BMY_HPP2 phosphatase geneor protein in a gene therapy regimen, as described herein, for thediagnoses, prognoses, treatment, amelioration, and/or prevention ofdiseases and/or disorders where a kinase activity is overly high, suchas a cancer where a kinase oncogene product has excessive activity orexcessive expression.

The present invention also encompasses the use of catalytically inactivevariants of BMY_HPP2 proteins, including fragments thereof, such as aprotein therapeutic, or the use of the encoding polynucleotide sequenceor as gene therapy, for example, in the diagnoses, prognosis, treatment,amelioration, and/or prevention of diseases or disorders wherephosphatase activity is overly high.

The present invention encompasses the use of antibodies directed againstthe BMY_HPP2 polypeptides, including fragment and/or variants thereof,of the present invention in diagnostics, as a biomarkers, and/or as atherapeutic agents.

The present invention encompasses the use of an inactive, non-catalytic,mutant of the BMY_HPP2 phosphatase as a substrate trapping mutant tobind cellular phosphoproteins or a library of phosphopeptides toidentify substrates of the BMY_HPP2 polypeptides.

The present invention encompasses the use of the BMY_HPP2 polypeptides,to identify inhibitors or activators of the BMY_HPP2 phosphataseactivity using either in vitro or ‘virtual’ (in silico) screeningmethods.

One embodiment of the invention relates to a method for identifying acompound as an activator or inhibitor of the BMY_HPP2 phosphatasecomprising the steps of: i.) contacting a BMY_HPP2 phosphatase inhibitoror activator labeled with an analytically detectable reagent with theBMY_HPP2 phosphatase under conditions sufficient to form a complex withthe inhibitor or activator; ii.) contacting said complex with a samplecontaining a compound to be identified; iii) and identifying thecompound as an inhibitor or activator by detecting the ability of thetest compound to alter the amount of labeled known BMY_HPP2 phosphataseinhibitor or activator in the complex.

Another embodiment of the invention relates to a method for identifyinga compound as an activator or inhibitor of a BMY_HPP2 phosphatasecomprising the steps of: i.) contacting the BMY_HPP2 phosphatase with acompound to be identified; and ii.) and measuring the ability of theBMY_HPP2 phosphatase to remove phosphate from a substrate.

The present invention also encomposses a method for identifying a ligandfor the BMY_HPP2 phosphatase comprising the steps of: i.) contacting theBMY_HPP2 phosphatase with a series of compounds under conditions topermit binding; and ii.) detecting the presence of any ligand-boundprotein.

Preferably, the above referenced methods comprise the BMY_HPP2phosphatase in a form selected from the group consisting of whole cells,cytosolic cell fractions, membrane cell fractions, purified or partiallypurified forms. The invention also relates to recombinantly expressedBMY_HPP2 phosphatase in a purified, substantially purified, orunpurified state. The invention further relates to BMY_HPP2 phosphatasefused or conjugated to a protein, peptide, or other molecule or compoundknown in the art, or referenced herein.

The present invention also encompasses pharmaceutical composition of theBMY_HPP2 phosphatase polypeptide comprising a compound identified byabove referenced methods and a pharmaceutically acceptable carrier.

Expression profiling designed to measure the steady state mRNA levelsencoding the BMY_HPP2 polypeptide showed predominately high expressionlevels in liver and kidney; to a significant extent, in the spleen, andto a lesser extent, in lung, testis, heart, intestine, pancreas, lymphnode, spinal cord, and prostate (as shown in FIG. 23).

Moreover, BLAST2 searches of the LifeSeq database (IncytePharmaceuticals) using the full-length BMY_HPP2 polynucleotide sequence(SEQ ID NO:151) led to the determination that the BMY_HPP2 sequence isexpressed significantly in lung libraries which include patients withemphysema and other pulmonary diseases. The BMY_HPP2 polynucleotide wasalso found to be expressed in aorta and endothelial cells stimulatedwith IL-1 and TNF-alpha. These findings suggest a potential involvementof the BMY_HPP2 polynucleotides and polypeptides in the incidence ofpulmonary disease and upregulation by IL-1 and TNF-alpha.

In addition, expanded expression profiling of the BMY_HPP2 polypeptidein normal tissues showed the highest level of expression in the adrenalgland, with lower but significant expression in the pineal pituitaryglands suggesting a role for modulators of BMY_HPP2 activity in thetreatment of endocrine disorders (as shown in FIG. 30). Consistent withthe expression pattern in lung libraries from the Incyte database above,high relative levels of expression were also seen in the parenchyma andbronchi of the lung, suggesting a role for modulators of BMY_HPP2activity in the treatment of respiratory diseases such as asthma orCOPD; in the kidney, suggesting a role for modulators of BMY_HPP2activity in the treatment of kidney disorders; in the liver, suggestinga role for modulators of BMY_HPP2 activity in the treatment of liverdisorders such as hepatitis or cirrhosis; in blood vessels from thechoroid plexus, coronary artery and pulmonary artery, suggesting a rolefor modulators of BMY_HPP2 activity in the treatment of circulatorydisorders such as hypertension; and in the nucleus accumbens of thebrain, suggesting a role for modulators of BMY_HPP2 activity in thetreatment of affective disorders such as bipolar disorder, schizophreniaand depression. In addition, the BMY_HPP2 was highly expressed in thetrachea, breast and uterus and significantly expressed in many othertissues within the human body.

The strong homology to phosphatases, particularly dual-specificityphosphatases, combined with the predominate localized expression inadrenal gland tissue suggests the human BMY_HPP2 phosphatasepolynucleotides and polypeptides, including agonists, antagonists,and/or fragments thereof, may be useful for treating, diagnosing,prognosing, amerliorating, and/or preventing endocrine disorders, whichinclude, but are not limited to adrenocortical hyperfunction,adrenocortical hypofunction, lethargy, Congenital adrenal hyperplasia,aberrant ACTH regulation, aberrant adrenaline regulation, disordersassociated with defects in P450C21, P450C18, P450C17, and P450C11hydroxylases and in 3-hydroxysteroid dehydrogenase (3-HSD), hirsutism,oligomenorrhea, acne, virilization, oligomenorrhea, femalepseudohermaphroditism, disorders associated with the incidence ofaberrant sexual characterisitics, disorders associated with aberrantcortisol secretion, hypertension, hypokalemia, hypogonadism, disordersassociated with aberrant androgen secretion, adrenal virilism, Adrenaladenomas, Adrenal carcinomas, disorders associated with aberrantaldosterone secretion, aldosteronism, disorders associated with aberrantsteriod biosynthesis, disorders associated with aberrant steriodtransport, disorders associated with aberrant steriod secretion,disorders associated with aberrant steriod excretion, Addison'ssyndrome, and Cushing's syndrome.

The strong homology to phosphatases, particularly dual-specificityphosphatases, combined with the significant expression in liverindicates the BMY_HPP2 polynucleotides and polypeptides, in addition to,fragments and variants thereof, would be useful for the detection andtreatment of liver disorders and cancers. Representative uses aredescribed in the “Hyperproliferative Disorders”, “Infectious Disease”,and “Binding Activity” sections below, and elsewhere herein. Briefly,the protein can be used for the detection, treatment, amelioration,and/or prevention of hepatoblastoma, jaundice, hepatitis, livermetabolic diseases and conditions that are attributable to thedifferentiation of hepatocyte progenitor cells, cirrhosis, hepaticcysts, pyrogenic abscess, amebic abcess, hydatid cyst,cystadenocarcinoma, adenoma, focal nodular hyperplasia, hemangioma,hepatocellulae carcinoma, cholangiocarcinoma, and angiosarcoma,granulomatous liver disease, liver transplantation, hyperbilirubinemia,jaundice, parenchymal liver disease, portal hypertension, hepatobiliarydisease, hepatic parenchyma, hepatic fibrosis, anemia, gallstones,cholestasis, carbon tetrachloride toxicity, beryllium toxicity, vinylchloride toxicity, choledocholithiasis, hepatocellular necrosis,aberrant metabolism of amino acids, aberrant metabolism ofcarbohydrates, aberrant synthesis proteins, aberrant synthesis ofglycoproteins, aberrant degradation of proteins, aberrant degradation ofglycoproteins, aberrant metabolism of drugs, aberrant metabolism ofhormones, aberrant degradation of drugs, aberrant degradation of drugs,aberrant regulation of lipid metabolism, aberrant regulation ofcholesterol metabolism, aberrant glycogenesis, aberrant glycogenolysis,aberrant glycolysis, aberrant gluconeogenesis, hyperglycemia, glucoseintolerance, hyperglycemia, decreased hepatic glucose uptake, decreasedhepatic glycogen synthesis, hepatic resistance to insulin,portal-systemic glucose shunting, peripheral insulin resistance,hormonal abnormalities, increased levels of systemic glucagon, decreasedlevels of systemic cortisol, increased levels of systemic insulin,hypoglycemia, decreased gluconeogenesis, decreased hepatic glycogencontent, hepatic resistance to glucagon, elevated levels of systemicaromatic amino acids, decreased levels of systemic branched-chain aminoacids, hepatic encephalopathy, aberrant hepatic amino acidtransamination, aberrant hepatic amino acid oxidative deamination,aberrant ammonia synthesis, aberant albumin secretion, hypoalbuminemia,aberrant cytochromes b5 function, aberrant P450 function, aberrantglutathione S-acyltransferase function, aberrant cholesterol synthesis,and aberrant bile acid synthesis.

Moreover, polynucleotides and polypeptides, including fragments,agonists and/or antagonists thereof, have uses which include, directlyor indirectly, treating, preventing, diagnosing, and/or prognosing thefollowing, non-limiting, hepatic infections: liver disease caused bysepsis infection, liver disease caused by bacteremia, liver diseasecaused by Pneomococcal pneumonia infection, liver disease caused byToxic shock syndrome, liver disease caused by Listeriosis, liver diseasecaused by Legionnaries' disease, liver disease caused by Brucellosisinfection, liver disease caused by Neisseria gonorrhoeae infection,liver disease caused by Yersinia infection, liver disease caused bySalmonellosis, liver disease caused by Nocardiosis, liver disease causedby Spirochete infection, liver disease caused by Treponema palliduminfection, liver disease caused by Brrelia burgdorferi infection, liverdisease caused by Leptospirosis, liver disease caused by Coxiellaburnetii infection, liver disease caused by Rickettsia richettsiiinfection, liver disease caused by Chlamydia trachomatis infection,liver disease caused by Chlamydia psittaci infection, liver diseasecaused by hepatitis virus infection, liver disease caused byEpstein-Barr virus infection in addition to any other hepatic diseaseand/or disorder implicated by the causative agents listed above orelsewhere herein.

The strong homology to dual specificity phophatases, combined with thelocalized expression in kidney tissue suggests the BMY_HPP2polynucleotides and polypeptides may be useful in treating, diagnosing,prognosing, and/or preventing renal diseases and/or disorders, whichinclude, but are not limited to: nephritis, renal failure, nephroticsyndrome, urinary tract infection, hematuria, proteinuria, oliguria,polyuria, nocturia, edema, hypertension, electrolyte disorders, sterilepyuria, renal osteodystrophy, large kidneys, renal transport defects,nephrolithiasis, azotemia, anuria, urinary retention, slowing of urinarystream, large prostate, flank tenderness, full bladder sensation aftervoiding, enuresis, dysuria, bacteriuria, kideny stones,glomerulonephritis, vasculitis, hemolytic uremic syndromes, thromboticthrombocytopenic purpura, malignant hypertension, casts,tubulointerstitial kidney diseases, renal tubular acidosis,pyelonephritis, hydronephritis, nephrotic syndrome, crush syndrome,and/or renal colic, in addition to Wilm's Tumor Disease, and congenitalkidney abnormalities such as horseshoe kidney, polycystic kidney, andFalconi's syndrome, for example.

The strong homology to dual specificity phosphatases, combined with thelocalized expression in spleen tissue, in addition to the expression inendothelial cells stimulated with IL-1 and TNF-alpha, suggests theBMY_HPP2 polynucleotides and polypeptides may be useful in treating,diagnosing, prognosing, and/or preventing immune diseases and/ordisorders. Representative uses are described in the “Immune Activity”,“Chemotaxis”, and “Infectious Disease” sections below, and elsewhereherein. Briefly, the strong expression in immune tissue indicates a rolein regulating the proliferation; survival; differentiation; and/oractivation of hematopoietic cell lineages, including blood stem cells.

The BMY_HPP2 polypeptide may also be useful as a preventative agent forimmunological disorders including arthritis, asthma, immunodeficiencydiseases such as AIDS, leukemia, rheumatoid arthritis, granulomatousdisease, inflammatory bowel disease, sepsis, acne, neutropenia,neutrophilia, psoriasis, hypersensitivities, such as T-cell mediatedcytotoxicity; immune reactions to transplanted organs and tissues, suchas host-versus-graft and graft-versus-host diseases, or autoimmunitydisorders, such as autoimmune infertility, lense tissue injury,demyelination, systemic lupus erythematosis, drug induced hemolyticanemia, rheumatoid arthritis, Sjogren's disease, and scleroderma. TheBMY_HPP2 polypeptide may be useful for modulating cytokine production,antigen presentation, or other processes, such as for boosting immuneresponses, etc.

Moreover, the protein may represent a secreted factor that influencesthe differentiation or behavior of other blood cells, or that recruitshematopoietic cells to sites of injury. Thus, this gene product isthought to be useful in the expansion of stem cells and committedprogenitors of various blood lineages, and in the differentiation and/orproliferation of various cell types. Furthermore, the protein may alsobe used to determine biological activity, raise antibodies, as tissuemarkers, to isolate cognate ligands or receptors, to identify agentsthat modulate their interactions, in addition to its use as anutritional supplement. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

The significant expression of BMY_HPP2 transcripts in lung libraries asobserved from electronic Northern's from the Incyte LifeSeq databasesuggests the potential utility for BMY_HPP2 polynucleotides andpolypeptides in treating, diagnosing, prognosing, and/or preventingpulmonary diseases and disorders which include the following, notlimiting examples: ARDS, emphysema, cystic fibrosis, interstitial lungdisease, chronic obstructive pulmonary disease, bronchitis,lymphangioleiomyomatosis, pneumonitis, eosinophilic pneumonias,granulomatosis, pulmonary infarction, pulmonary fibrosis,pneumoconiosis, alveolar hemorrhage, neoplasms, lung abscesses, empyema,and increased susceptibility to lung infections (e.g.,immumocompromised, HIV, etc.), for example.

Moreover, polynucleotides and polypeptides, including fragments,agonists and/or antagonists thereof, have uses which include, directlyor indirectly, treating, preventing, diagnosing, and/or prognosing thefollowing, non-limiting, pulmonary infections: pnemonia, bacterialpnemonia, viral pnemonia (for example, as caused by Influenza virus,Respiratory syncytial virus, Parainfluenza virus, Adenovirus,Coxsackievirus, Cytomegalovirus, Herpes simplex virus, Hantavirus,etc.), mycobacteria pnemonia (for example, as caused by Mycobacteriumtuberculosis, etc.) mycoplasma pnemonia, fungal pnemonia (for example,as caused by Pneumocystis carinii, Histoplasma capsulatum, Coccidioidesimmitis, Blastomyces dermatitidis, Candida sp., Cryptococcus neoformans,Aspergillus sp., Zygomycetes, etc.), Legionnaires' Disease, Chlamydiapnemonia, aspiration pnemonia, Nocordia sp. Infections, parasiticpnemonia (for example, as caused by Strongyloides, Toxoplasma gondii,etc.) necrotizing pnemonia, in addition to any other pulmonary diseaseand/or disorder (e.g., non-pneumonia) implicated by the causative agentslisted above or elsewhere herein.

Antisense oligonucleotides directed against BMY_HPP2 provided evidencesuggesting its involvement in the regulation of mammalian cell cycleprogression (see Example 56). Subjecting cells with an effective amountof a pool of five antisense oligoncleotides resulted in a significantincrease in Cyclin D expression/activity providing convincing evidencethat BMY_HPP2 at least regulates the activity and/or expression ofCyclin D either directly, or indirectly. Moreover, the results suggestthe physiological role of BMY_HPP2 is the negative regulation of CyclinD activity and/or expression, either directly or indirectly.

In preferred embodiments, BMY_HPP2 polynucleotides and polypeptides,including fragments thereof, are useful for treating, diagnosing, and/orameliorating cell cycle defects, disorders related to aberrantphosphorylation, disorders related to aberrant signal transduction,proliferating disorders, and/or cancers.

Moreover, BMY_HPP2 polynucleotides and polypeptides, including fragmentsthereof, are useful for decreasing cellular proliferation, decreasingcellular proliferation in rapidly proliferating cells, increasing thenumber of cells in the G1 phase of the cell cycle, and decreasing thenumber of cells that progress to the S phase of the cell cycle.

In preferred embodiments, agonists directed to BMY_HPP2 are useful fordecreasing cellular proliferation, decreasing cellular proliferation inrapidly proliferating cells, increasing the number of cells in the G1phase of the cell cycle, and decreasing the number of cells thatprogress to the S phase of the cell cycle.

Moreover, antagonists directed against BMY_HPP2 are useful forincreasing cellular proliferation, increasing cellular proliferation inrapidly proliferating cells, decreasing the number of cells in the G1phase of the cell cycle, and increasing the number of cells thatprogress to the S phase of the cell cycle. Such antagonists would beparticularly useful for transforming normal cells into immortalized celllines, stimulating hematopoietic cells to grow and divide, increasingrecovery rates of cancer patients that have undergone chemotherapy orother therapeutic regimen, by boosting their immune responses, etc.

The BMY_HPP2 polypeptide has been shown to comprise one glycosylationsites according to the Motif algorithm (Genetics Computer Group, Inc.).As discussed more specifically herein, protein glycosylation is thoughtto serve a variety of functions including: augmentation of proteinfolding, inhibition of protein aggregation, regulation of intracellulartrafficking to organelles, increasing resistance to proteolysis,modulation of protein antigenicity, and mediation of intercellularadhesion.

Asparagine glycosylation sites have the following consensus pattern,N-{P}-[ST]-{P}, wherein N represents the glycosylation site. However, itis well known that that potential N-glycosylation sites are specific tothe consensus sequence Asn-Xaa-Ser/Thr. However, the presence of theconsensus tripeptide is not sufficient to conclude that an asparagineresidue is glycosylated, due to the fact that the folding of the proteinplays an important role in the regulation of N-glycosylation. It hasbeen shown that the presence of proline between Asn and Ser/Thr willinhibit N-glycosylation; this has been confirmed by a recent statisticalanalysis of glycosylation sites, which also shows that about 50% of thesites that have a proline C-terminal to Ser/Thr are not glycosylated.Additional information relating to asparagine glycosylation may be foundin reference to the following publications, which are herebyincorporated by reference herein: Marshall R. D., Annu. Rev. Biochem.41:673-702 (1972); Pless D. D., Lennarz W. J., Proc. Natl. Acad. Sci.U.S.A. 74:134-138 (1977); Bause E., Biochem. J. 209:331-336 (1983);Gavel Y., von Heijne G., Protein Eng. 3:433-442 (1990); and Miletich J.P., Broze G. J. Jr., J. Biol. Chem. 265:11397-11404 (1990).

In preferred embodiments, the following asparagine glycosylation sitepolypeptide is encompassed by the present invention: GVQPPNFSWVLPGR (SEQID NO:164). Polynucleotides encoding this polypeptide are also provided.The present invention also encompasses the use of this BMY_HPP2asparagine glycosylation site polypeptide as an immunogenic and/orantigenic epitope as described elsewhere herein.

The BMY_HPP2 polypeptides of the present invention were determined tocomprise several phosphorylation sites based upon the Motif algorithm(Genetics Computer Group, Inc.). The phosphorylation of such sites mayregulate some biological activity of the BMY_HPP2 polypeptide. Forexample, phosphorylation at specific sites may be involved in regulatingthe proteins ability to associate or bind to other molecules (e.g.,proteins, ligands, substrates, DNA, etc.). In the present case,phosphorylation may modulate the ability of the BMY_HPP2 polypeptide toassociate with other potassium channel alpha subunits, beta subunits, orits ability to modulate potassium channel function.

The BMY_HPP2 polypeptide was predicted to comprise one PKCphosphorylation site using the Motif algorithm (Genetics Computer Group,Inc.). In vivo, protein kinase C exhibits a preference for thephosphorylation of serine or threonine residues. The PKC phosphorylationsites have the following consensus pattern: [ST]-x-[RK], where S or Trepresents the site of phosphorylation and ‘x’ an intervening amino acidresidue. Additional information regarding PKC phosphorylation sites canbe found in Woodget J. R., Gould K. L., Hunter T., Eur. J. Biochem.161:177-184 (1986), and Kishimoto A., Nishiyama K., Nakanishi H.,Uratsuji Y., Nomura H., Takeyama Y., Nishizuka Y., J. Biol. Chem.260:12492-12499 (1985); which are hereby incorporated by referenceherein.

In preferred embodiments, the following PKC phosphorylation sitepolypeptide is encompassed by the present invention: HLVSLTERGPPHS (SEQID NO:165). Polynucleotides encoding these polypeptides are alsoprovided. The present invention also encompasses the use of theseBMY_HPP2 PKC phosphorylation site polypeptides as immunogenic and/orantigenic epitopes as described elsewhere herein.

In further confirmation of the human BMY_HPP2 polypeptide representing anovel human phosphatase polypeptide, the BMY_HPP2 polypeptide has beenshown to comprise a tyrosine specific protein phosphatase active sitedomain according to the Motif algorithm (Genetics Computer Group, Inc.).

Tyrosine specific protein phosphatases (EC 3.1.3.48) (PTPase) areenzymes that catalyze the removal of a phosphate group attached to atyrosine residue. These enzymes are very important in the control ofcell growth, proliferation, differentiation and transformation. Multipleforms of PTPase have been characterized and can be classified into twocategories: soluble PTPases and transmembrane receptor proteins thatcontain PTPase domain(s).

The currently known PTPases are listed below: Soluble PTPases, PTPN1(PTP-1B), PTPN2 (T-cell PTPase; TC-PTP), PTPN3 (H1) and PTPN4 (MEG),enzymes that contain an N-terminal band 4.1-like domain and could act atjunctions between the membrane and cytoskeleton, PTPN5 (STEP), PTPN6(PTP-1C; HCP; SHP) and PTPN11 (PTP-2C; SH-PTP3; Syp), enzymes whichcontain two copies of the SH2 domain at its N-terminal extremity (e.g.,the Drosophila protein corkscrew (gene csw) also belongs to thissubgroup), PTPN7 (LC-PTP; Hematopoietic protein-tyrosine phosphatase;HePTP), PTPN8 (70Z-PEP), PTPN9 (MEG2), PTPN12 (PTP-G1; PTP-P19), YeastPTP1, Yeast PTP2 which may be involved in the ubiquitin-mediated proteindegradation pathway, Fission yeast pyp1 and pyp2 which play a role ininhibiting the onset of mitosis, Fission yeast pyp3 which contributes tothe dephosphorylation of cdc2, Yeast CDC14 which may be involved inchromosome segregation, Yersinia virulence plasmid PTPAses (gene yopH),Autographa californica nuclear polyhedrosis virus 19 Kd PTPase, Dualspecificity PTPases, DUSP1 (PTPN10; MAP kinase phosphatase-1; MKP-1);which dephosphorylates MAP kinase on both Thr-183 and Tyr-185, DUSP2(PAC-1), a nuclear enzyme that dephosphorylates MAP kinases ERK1 andERK2 on both Thr and Tyr residues, DUSP3 (VHR), DUSP4 (HVH2), DUSP5(HVH3), DUSP6 (Pyst1; MKP-3), DUSP7 (Pyst2; MKP-X), Yeast MSG5, a PTPasethat dephosphorylates MAP kinase FUS3, Yeast YVH1, Vaccinia virus H1PTPase—a dual specificity phosphatase,

Structurally, all known receptor PTPases, are made up of a variablelength extracellular domain, followed by a transmembrane region and aC-terminal catalytic cytoplasmic domain. Some of the receptor PTPasescontain fibronectin type III (FN-III) repeats, immunoglobulin-likedomains, MAM domains or carbonic anhydrase-like domains in theirextracellular region. The cytoplasmic region generally contains twocopies of the PTPAse domain. The first seems to have enzymatic activity,while the second is inactive but seems to affect substrate specificityof the first. In these domains, the catalytic cysteine is generallyconserved but some other, presumably important, residues are not.

PTPase domains consist of about 300 amino acids. There are two conservedcysteines, the second one has been shown to be absolutely required foractivity. Furthermore, a number of conserved residues in its immediatevicinity have also been shown to be important.

A consensus sequence for tyrosine specific protein phophatases isprovided as follows:

[LIVMF]-H-C-x(2)-G-x(3)-[STC]-[STAGP]-x-[LIVMFY], wherein C is theactive site residue and “X” represents any amino acid.

Additional information related to tyrosine specific protein phosphatasedomains and proteins may be found in reference to the followingpublications Fischer E. H., Charbonneau H., Tonks N. K., Science253:401-406 (1991); Charbonneau H., Tonks N. K., Annu. Rev. Cell Biol.8:463-493 (1992); Trowbridge I. S., J. Biol. Chem. 266:23517-23520(1991); Tonks N. K., Charbonneau H., Trends Biochem. Sci. 14:497-500(1989); and Hunter T., Cell 58:1013-1016 (1989); which are herebyincorporated herein by reference in their entirety.

In preferred embodiments, the following tyrosine specific proteinphosphatase active site domain polypeptide is encompassed by the presentinvention: GEAVGVHCALGFGRTGTMLACYL (SEQ ID NO:166). Polynucleotidesencoding these polypeptides are also provided. The present inventionalso encompasses the use of this tyrosine specific protein phosphataseactive site domain polypeptide as an immunogenic and/or antigenicepitope as described elsewhere herein.

In preferred embodiments, the following N-terminal BMY_HPP2 deletionpolypeptides are encompassed by the present invention: M1-K150, G2-K150,V3-K150, Q4-K150, P5-K150, P6-K150, N7-K150, F8-K150, S9-K150, W10-K150,V11-K150, L12-K150, P13-K150, G14-K150, R15-K150, L16-K150, A17-K150,G18-K150, L19-K150, A20-K150, L21-K150, P22-K150, R23-K150, L24-K150,P25-K150, A26-K150, H27-K150, Y28-K150, Q29-K150, F30-K150, L31-K150,L32-K150, D33-K150, L34-K150, G35-K150, V36-K150, R37-K150, H38-K150,L39-K150, V40-K150, S41-K150, L42-K150, T43-K150, E44-K150, R45-K150,G46-K150, P47-K150, P48-K150, H49-K150, S50-K150, D51-K150, S52-K150,C53-K150, P54-K150, G55-K150, L56-K150, T57-K150, L58-K150, H59-K150,R60-K150, L61-K150, R62-K150, I63-K150, P64-K150, D65-K150, F66-K150,C67-K150, P68-K150, P69-K150, A70-K150, P71-K150, D72-K150, Q73-K150,I74-K150, D75-K150, R76-K150, F77-K150, V78-K150, Q79-K150, I80-K150,V81-K150, D82-K150, E83-K150, A84-K150, N85-K150, A86-K150, R87-K150,G88-K150, E89-K150, A90-K150, V91-K150, G92-K150, V93-K150, H94-K150,C95-K150, A96-K150, L97-K150, G98-K150, F99-K150, G100-K150, R101-K150,T102-K150, G103-K150, T104-K150, M105-K150, L106-K150, A107-K150,C108-K150, Y109-K150, L110-K150, V111-K150, K112-K150, E113-K150,R114-K150, G115-K150, L116-K150, A117-K150, A118-K150, G119-K150,D120-K150, A121-K150, I122-K150, A123-K150, E124-K150, I125-K150,R126-K150, R127-K150, L128-K150, R129-K150, P130-K150, G131-K150,S132-K150, I133-K150, E134-K150, T135-K150, Y136-K150, E137-K150,Q138-K150, E139-K150, K140-K150, A141-K150, V142-K150, F143-K150, and/orQ144-K150 of SEQ ID NO:152. Polynucleotide sequences encoding thesepolypeptides are also provided. The present invention also encompassesthe use of these N-terminal BMY_HPP2 deletion polypeptides asimmunogenic and/or antigenic epitopes as described elsewhere herein.

In preferred embodiments, the following C-terminal BMY_HPP2 deletionpolypeptides are encompassed by the present invention: M1-K150, M1-T149,M1-R148, M1-Q147, M1-Y146, M1-F145, M1-Q144, M1-F143, M1-V142, M1-A141,M1-K140, M1-E139, M1-Q138, M1-E137, M1-Y136, M1-T135, M1-E134, M1-I133,M1-S132, M1-G131, M1-P130, M1-R129, M1-L128, M1-R127, M1-R126, M1-I125,M1-E124, M1-A123, M1-I122, M1-A121, M1-D120, M1-G119, M1-A118, M1-A117,M1-L116, M1-G115, M1-R114, M1-E113, M1-K112, M1-V111, M1-L110, M1-Y109,M1-C108, M1-A107, M1-L106, M1-M105, M1-T104, M1-G103, M1-T102, M1-R101,M1-G100, M1-F99, M1-G98, M1-L97, M1-A96, M1-C95, M1-H94, M1-V93, M1-G92,M1-V91, M1-A90, M1-E89, M1-G88, M1-R87, M1-A86, M1-N85, M1-A84, M1-E83,M1-D82, M1-V81, M1-I80, M1-Q79, M1-V78, M1-F77, M1-R76, M1-D75, M1-I74,M1-Q73, M1-D72, M1-P71, M1-A70, M1-P69, M1-P68, M1-C67, M1-F66, M1-D65,M1-P64, M1-I63, M1-R62, M1-L61, M1-R60, M1-H59, M1-L58, M1-T57, M1-L56,M1-G55, M1-P54, M1-C53, M1-S52, M1-D51, M1-S50, M1-H49, M1-P48, M1-P47,M1-G46, M1-R45, M1-E44, M1-T43, M1-L42, M1-S41, M1-V40, M1-L39, M1-H38,M1-R37, M1-V36, M1-G35, M1-L34, M1-D33, M1-L32, M1-L31, M1-F30, M1-Q29,M1-Y28, M1-H27, M1-A26, M1-P25, M1-L24, M1-R23, M1-P22, M1-L21, M1-A20,M1-L19, M1-G18, M1-A17, M1-L16, M1-R15, M1-G14, M1-P13, M1-L12, M1-V11,M1-W10, M1-S9, M1-F8, and/or M1-N7 of SEQ ID NO:152. Polynucleotidesequences encoding these polypeptides are also provided. The presentinvention also encompasses the use of these C-terminal BMY_HPP2 deletionpolypeptides as immunogenic and/or antigenic epitopes as describedelsewhere herein.

In preferred embodiments, the following BMY_HPP2 phosphatase active sitedomain amino acid substitutions are encompassed by the presentinvention: wherein M1 is substituted with either an A, C, D, E, F, G, H,I, K, L, N, P, Q, R, S, T, V, W, or Y; wherein G2 is substituted witheither an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;wherein V3 is substituted with either an A, C, D, E, F, G, H, I, K, L,M, N, P, Q, R, S, T, W, or Y; wherein Q4 is substituted with either anA, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein P5is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R,S, T, V, W, or Y; wherein P6 is substituted with either an A, C, D, E,F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein N7 issubstituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S,T, V, W, or Y; wherein F8 is substituted with either an A, C, D, E, G,H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein S9 is substitutedwith either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, orY; wherein W10 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, N, P, Q, R, S, T, V, or Y; wherein V11 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; whereinL12 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q,R, S, T, V, W, or Y; wherein P13 is substituted with either an A, C, D,E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein G14 issubstituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S,T, V, W, or Y; wherein R15 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein L16 is substitutedwith either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, orY; wherein A17 is substituted with either a C, D, E, F, G, H, I, K, L,M, N, P, Q, R, S, T, V, W, or Y; wherein G18 is substituted with eitheran A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; whereinL19 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q,R, S, T, V, W, or Y; wherein A20 is substituted with either a C, D, E,F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L21 issubstituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S,T, V, W, or Y; wherein P22 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein R23 is substitutedwith either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, orY; wherein L24 is substituted with either an A, C, D, E, F, G, H, I, K,M, N, P, Q, R, S, T, V, W, or Y; wherein P25 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; whereinA26 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q,R, S, T, V, W, or Y; wherein H27 is substituted with either an A, C, D,E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Y28 issubstituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, T, V, or W; wherein Q29 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein F30 is substitutedwith either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, orY; wherein L31 is substituted with either an A, C, D, E, F, G, H, I, K,M, N, P, Q, R, S, T, V, W, or Y; wherein L32 is substituted with eitheran A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; whereinD33 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q,R, S, T, V, W, or Y; wherein L34 is substituted with either an A, C, D,E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein G35 issubstituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S,T, V, W, or Y; wherein V36 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein R37 is substitutedwith either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, orY; wherein H38 is substituted with either an A, C, D, E, F, G, I, K, L,M, N, P, Q, R, S, T, V, W, or Y; wherein L39 is substituted with eitheran A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; whereinV40 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P,Q, R, S, T, W, or Y; wherein S41 is substituted with either an A, C, D,E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein L42 issubstituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S,T, V, W, or Y; wherein T43 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein E44 is substitutedwith either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, orY; wherein R45 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, N, P, Q, S, T, V, W, or Y; wherein G46 is substituted with eitheran A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; whereinP47 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q,R, S, T, V, W, or Y; wherein P48 is substituted with either an A, C, D,E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein H49 issubstituted with either an A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S,T, V, W, or Y; wherein S50 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein D51 is substitutedwith either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, orY; wherein S52 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, N, P, Q, R, T, V, W, or Y; wherein C53 is substituted with eitheran A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; whereinP54 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q,R, S, T, V, W, or Y; wherein G55 is substituted with either an A, C, D,E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L56 issubstituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S,T, V, W, or Y; wherein T57 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein L58 is substitutedwith either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, orY; wherein H59 is substituted with either an A, C, D, E, F, G, I, K, L,M, N, P, Q, R, S, T, V, W, or Y; wherein R60 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; whereinL61 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q,R, S, T, V, W, or Y; wherein R62 is substituted with either an A, C, D,E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein I63 issubstituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S,T, V, W, or Y; wherein P64 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein D65 is substitutedwith either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, orY; wherein F66 is substituted with either an A, C, D, E, G, H, I, K, L,M, N, P, Q, R, S, T, V, W, or Y; wherein C67 is substituted with eitheran A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; whereinP68 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q,R, S, T, V, W, or Y; wherein P69 is substituted with either an A, C, D,E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein A70 issubstituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,T, V, W, or Y; wherein P71 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein D72 is substitutedwith either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, orY; wherein Q73 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, N, P, R, S, T, V, W, or Y; wherein I74 is substituted with eitheran A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; whereinD75 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q,R, S, T, V, W, or Y; wherein R76 is substituted with either an A, C, D,E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein F77 issubstituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S,T, V, W, or Y; wherein V78 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein Q79 is substitutedwith either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, orY; wherein I80 is substituted with either an A, C, D, E, F, G, H, K, L,M, N, P, Q, R, S, T, V, W, or Y; wherein V81 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; whereinD82 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q,R, S, T, V, W, or Y; wherein E83 is substituted with either an A, C, D,F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A84 issubstituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,T, V, W, or Y; wherein N85 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein A86 is substitutedwith either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, orY; wherein R87 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, N, P, Q, S, T, V, W, or Y; wherein G88 is substituted with eitheran A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; whereinE89 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q,R, S, T, V, W, or Y; wherein A90 is substituted with either a C, D, E,F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V91 issubstituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, T, W, or Y; wherein G92 is substituted with either an A, C, D, E, F,H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V93 is substitutedwith either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, orY; wherein H94 is substituted with either an A, C, D, E, F, G, I, K, L,M, N, P, Q, R, S, T, V, W, or Y; wherein C95 is substituted with eitheran A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; whereinA96 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q,R, S, T, V, W, or Y; wherein L97 is substituted with either an A, C, D,E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein G98 issubstituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S,T, V, W, or Y; wherein F99 is substituted with either an A, C, D, E, G,H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein G100 is substitutedwith either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, orY; wherein R101 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, N, P, Q, S, T, V, W, or Y; wherein T102 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; whereinG103 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P,Q, R, S, T, V, W, or Y; wherein T104 is substituted with either an A, C,D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein M105 issubstituted with either an A, C, D, E, F, G, H, I, K, L, N, P, Q, R, S,T, V, W, or Y; wherein L106 is substituted with either an A, C, D, E, F,G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein A107 is substitutedwith either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, orY; wherein C108 is substituted with either an A, D, E, F, G, H, I, K, L,M, N, P, Q, R, S, T, V, W, or Y; wherein Y109 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; whereinL110 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P,Q, R, S, T, V, W, or Y; and/or wherein V111 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y of SEQ IDNO:152, in addition to any combination thereof. The present inventionalso encompasses the use of these BMY_HPP2 phosphatase active sitedomain amino acid substituted polypeptides as immunogenic and/orantigenic epitopes as described elsewhere herein.

In preferred embodiments, the following BMY_HPP2 phosphatase active sitedomain conservative amino acid substitutions are encompassed by thepresent invention: wherein M1 is substituted with either an A, G, S, orT; wherein G2 is substituted with either an A, M, S, or T; wherein V3 issubstituted with either an A, I, or L; wherein Q4 is substituted with aN; wherein P5 is a P; wherein P6 is a P; wherein N7 is substituted witha Q; wherein F8 is substituted with either a W, or Y; wherein S9 issubstituted with either an A, G, M, or T; wherein W10 is either an F, orY; wherein V11 is substituted with either an A, I, or L; wherein L12 issubstituted with either an A, I, or V; wherein P13 is a P; wherein G14is substituted with either an A, M, S, or T; wherein R15 is substitutedwith either a K, or H; wherein L16 is substituted with either an A, I,or V; wherein A17 is substituted with either a G, I, L, M, S, T, or V;wherein G18 is substituted with either an A, M, S, or T; wherein L19 issubstituted with either an A, I, or V; wherein A20 is substituted witheither a G, I, L, M, S, T, or V; wherein L21 is substituted with eitheran A, I, or V; wherein P22 is a P; wherein R23 is substituted witheither a K, or H; wherein L24 is substituted with either an A, I, or V;wherein P25 is a P; wherein A26 is substituted with either a G, I, L, M,S, T, or V; wherein H27 is substituted with either a K, or R; whereinY28 is either an F, or W; wherein Q29 is substituted with a N; whereinF30 is substituted with either a W, or Y; wherein L31 is substitutedwith either an A, I, or V; wherein L32 is substituted with either an A,I, or V; wherein D33 is substituted with an E; wherein L34 issubstituted with either an A, I, or V; wherein G35 is substituted witheither an A, M, S, or T; wherein V36 is substituted with either an A, I,or L; wherein R37 is substituted with either a K, or H; wherein H38 issubstituted with either a K, or R; wherein L39 is substituted witheither an A, I, or V; wherein V40 is substituted with either an A, I, orL; wherein S41 is substituted with either an A, G, M, or T; wherein L42is substituted with either an A, I, or V; wherein T43 is substitutedwith either an A, G, M, or S; wherein E44 is substituted with a D;wherein R45 is substituted with either a K, or H; wherein G46 issubstituted with either an A, M, S, or T; wherein P47 is a P; whereinP48 is a P; wherein H49 is substituted with either a K, or R; whereinS50 is substituted with either an A, G, M, or T; wherein D51 issubstituted with an E; wherein S52 is substituted with either an A, G,M, or T; wherein C53 is a C; wherein P54 is a P; wherein G55 issubstituted with either an A, M, S, or T; wherein L56 is substitutedwith either an A, I, or V; wherein T57 is substituted with either an A,G, M, or S; wherein L58 is substituted with either an A, I, or V;wherein H59 is substituted with either a K, or R; wherein R60 issubstituted with either a K, or H; wherein L61 is substituted witheither an A, I, or V; wherein R62 is substituted with either a K, or H;wherein I63 is substituted with either an A, V, or L; wherein P64 is aP; wherein D65 is substituted with an E; wherein F66 is substituted witheither a W, or Y; wherein C67 is a C; wherein P68 is a P; wherein P69 isa P; wherein A70 is substituted with either a G, I, L, M, S, T, or V;wherein P71 is a P; wherein D72 is substituted with an E; wherein Q73 issubstituted with a N; wherein I74 is substituted with either an A, V, orL; wherein D75 is substituted with an E; wherein R76 is substituted witheither a K, or H; wherein F77 is substituted with either a W, or Y;wherein V78 is substituted with either an A, I, or L; wherein Q79 issubstituted with a N; wherein I80 is substituted with either an A, V, orL; wherein V81 is substituted with either an A, I, or L; wherein D82 issubstituted with an E; wherein E83 is substituted with a D; wherein A84is substituted with either a G, I, L, M, S, T, or V; wherein N85 issubstituted with a Q; wherein A86 is substituted with either a G, I, L,M, S, T, or V; wherein R87 is substituted with either a K, or H; whereinG88 is substituted with either an A, M, S, or T; wherein E89 issubstituted with a D; wherein A90 is substituted with either a G, I, L,M, S, T, or V; wherein V91 is substituted with either an A, I, or L;wherein G92 is substituted with either an A, M, S, or T; wherein V93 issubstituted with either an A, I, or L; wherein H94 is substituted witheither a K, or R; wherein C95 is a C; wherein A96 is substituted witheither a G, I, L, M, S, T, or V; wherein L97 is substituted with eitheran A, I, or V; wherein G98 is substituted with either an A, M, S, or T;wherein F99 is substituted with either a W, or Y; wherein G100 issubstituted with either an A, M, S, or T; wherein R101 is substitutedwith either a K, or H; wherein T102 is substituted with either an A, G,M, or S; wherein G103 is substituted with either an A, M, S, or T;wherein T104 is substituted with either an A, G, M, or S; wherein M105is substituted with either an A, G, S, or T; wherein L106 is substitutedwith either an A, I, or V; wherein A107 is substituted with either a G,I, L, M, S, T, or V; wherein C108 is a C; wherein Y109 is either an F,or W; wherein L110 is substituted with either an A, I, or V; and/orwherein V111 is substituted with either an A, I, or L of SEQ ID NO:152in addition to any combination thereof. Other suitable substitutionswithin the BMY_HPP2 phosphatase active site domain are encompassed bythe present invention and are referenced elsewhere herein. The presentinvention also encompasses the use of these BMY_HPP2 phosphatase activesite domain conservative amino acid substituted polypeptides asimmunogenic and/or antigenic epitopes as described elsewhere herein.

In preferred embodiments, the present invention encompasses apolynucleotide lacking the initiating start codon, in addition to, theresulting encoded polypeptide of BMY_HPP2. Specifically, the presentinvention encompasses the polynucleotide corresponding to nucleotides 92thru 538 of SEQ ID NO:151, and the polypeptide corresponding to aminoacids 2 thru 150 of SEQ ID NO:152. Also encompassed are recombinantvectors comprising said encoding sequence, and host cells comprisingsaid vector.

The present invention also provides a three-dimensional homology modelof the BMY_HPP2 polypeptide (see FIG. 32) representing amino acidresidues M1 to K150 of the polypeptide sequence of BMY_HPP2 (amino acidresidues M1 to K150 of SEQ ID NO:152). A three-dimensional homologymodel can be constructed on the basis of the known structure of ahomologous protein (Greer et al, 1991, Lesk, et al, 1992, Cardozo, etal, 1995, Yuan, et al, 1995). The homology model of the BMY_HPP2polypeptide sequence (SEQ ID NO:152), was based upon the homologousstructure of 1vhr from the N-terminus of the human dual specificityphosphatase (vaccinia H1-related phosphatase VN1) (residues N31-K179;Protein Data Bank, PDB entry 1vhr chain A; Genbank Accession No.gi|1633321; SEQ ID NO:207) and is defined by the set of structuralcoordinates set forth in Table IX herein.

Homology models are useful when there is no experimental informationavailable on the protein of interest. A 3-dimensional model can beconstructed on the basis of the known structure of a homologous protein(Greer et al, 1991, Lesk, et al, 1992, Cardozo, et al, 1995, Sali, etal, 1995).

Those of skill in the art will understand that a homology model isconstructed on the basis of first identifying a template, or, protein ofknown structure which is similar to the protein without known structure.This can be accomplished by through pairwise alignment of sequencesusing such programs as FASTA (Pearson, et al 1990) and BLAST (Altschul,et al, 1990). In cases where sequence similarity is high (greater than30%) these pairwise comparison methods may be adequate. Likewise,multiple sequence alignments or profile-based methods can be used toalign a query sequence to an alignment of multiple (structurally andbiochemically) related proteins. When the sequence similarity is low,more advanced techniques are used such as fold recognition (proteinthreading; Hendlich, et al, 1990), where the compatibility of aparticular sequence with the 3-dimensional fold of a potential templateprotein is gauged on the basis of a knowledge-based potential. Followingthe initial sequence alignment, the query template can be optimallyaligned by manual manipulation or by incorporation of other features(motifs, secondary structure predictions, and allowed sequenceconservation). Next, structurally conserved regions can be identifiedand used to construct the core secondary structure (Sali, et al, 1995).Loops can be added using knowledge-based techniques, and refinedperforming forcefield calculations (Sali, et al, 1995, Cardozo, et al,1995).

For BMY_HPP2 the pairwise alignment method FASTA (Pearson, et al 1990)and fold recognition methods (protein threading) generated identicalsequence alignments for a portion (residues M1 to K150 of SEQ ID NO:152)of BMY_HPP2 aligned with the sequence of 1vhr from the N-terminus of thehuman dual specificity phosphatase (vaccinia H1-related phosphatase VN1)(residues N31-K179; Protein Data Bank, PDB entry 1vhr chain A; GenbankAccession No. gi|1633321; SEQ ID NO:207). The alignment of BMY-HPP2 withPDB entry 1vhr is set forth in FIG. 31. In this invention, the homologymodel of BMY_HPP2 was derived from the sequence alignment set forth inFIG. 31, and hence an overall atomic model including plausible sidechainorientations using the program LOOK (Levitt, 1992). The threedimensional model for BMY-HPP2 is defined by the set of structurecoordinates as set forth in Table IX and visualized in FIG. 32.

In order to recognize errors in three-dimensional structures knowledgebased mean fields can be used to judge the quality of protein folds(Sippl 1993). The methods can be used to recognize misfolded structuresas well as faulty parts of structural models. The technique generates anenergy graph where the energy distribution for a given protein fold isdisplayed on the y-axis and residue position in the protein fold isdisplayed on the x-axis. The knowledge based mean fields compose a forcefield derived from a set of globular protein structures taken as asubset from the Protein Data Bank (Bernstein et. al. 1977). To analyzethe quality of a model the energy distribution is plotted and comparedto the energy distribution of the template from which the model wasgenerated. FIG. 33 shows the energy graph for the BMY_HPP2 model (dottedline) and the template (1vhr, a dual-specificity phosphatase) from whichthe model was generated. It is clear that the model and template havesimilar energies over the aligned region, suggesting that BMY_HPP2 is ina “native-like” conformation. This graph supports the motif and sequencealignments in confirming that the three dimensional structurecoordinates of BMY_HPP2 are an accurate and useful representation forthe polypeptide.

The term “structure coordinates” refers to Cartesian coordinatesgenerated from the building of a homology model.

Those of skill in the art will understand that a set of structurecoordinates for a protein is a relative set of points that define ashape in three dimensions. Thus, it is possible that an entirelydifferent set of coordinates could define a similar or identical shape.Moreover, slight variations in the individual coordinates, as emanatefrom generation of similar homology models using different alignmenttemplates (i.e., other than the structure coordinates of 1vhr), and/orusing different methods in generating the homology model, will haveminor effects on the overall shape. Variations in coordinates may alsobe generated because of mathematical manipulations of the structurecoordinates. For example, the structure coordinates set forth in TableIX and visualized in FIG. 32 could be manipulated by fractionalizationof the structure coordinates; integer additions or subtractions to setsof the structure coordinates, inversion of the structure coordinates orany combination of the above.

Various computational analyses are therefore necessary to determinewhether a molecule or a portion thereof is sufficiently similar to allor parts of BMY_HPP2 described above as to be considered the same. Suchanalyses may be carried out in current software applications, such asINSIGHTII (Molecular Simulations Inc., San Diego, Calif.) version 2000and as described in the accompanying User's Guide.

Using the superimposition tool in the program INSIGHTII comparisons canbe made between different structures and different conformations of thesame structure. The procedure used in INSIGHTII to compare structures isdivided into four steps: 1) load the structures to be compared; 2)define the atom equivalencies in these structures; 3) perform a fittingoperation; and 4) analyze the results. Each structure is identified by aname. One structure is identified as the target (i.e., the fixedstructure); the second structure (i.e., moving structure) is identifiedas the source structure. Since atom equivalency within INSIGHTII isdefined by user input, for the purpose of this invention we will defineequivalent atoms as protein backbone atoms (N, Cα, C and O) for allconserved residues between the two structures being compared. We willalso consider only rigid fitting operations. When a rigid fitting methodis used, the working structure is translated and rotated to obtain anoptimum fit with the target structure. The fitting operation uses analgorithm that computes the optimum translation and rotation to beapplied to the moving structure, such that the root mean squaredifference of the fit over the specified pairs of equivalent atom is anabsolute minimum. This number, given in angstroms, is reported byINSIGHTII. For the purpose of this invention, any homology model of aBMY_HPP2 that has a root mean square deviation of conserved residuebackbone atoms (N, Cα, C, O) of less than 3.0 A when superimposed on therelevant backbone atoms described by structure coordinates listed inTable IX and visualized in FIG. 32 are considered identical. Morepreferably, the root mean square deviation is less than 2.0 Å.

This invention as embodied by the homology model enables thestructure-based design of modulators of the biological function ofBMY_HPP2, as well as mutants with altered biological function and/orspecificity.

There is 23% sequence identity between catalytic domain of BMY_HPP2 andthe human dual specificity phosphatase VHR (Yuvaniyama, J., et al.,1996; PDB identifier 1vhr) which was used as the template for 3D modelgeneration as determined by the GAP program within GCG (GeneticsComputer Group, Wisconsin). For the BMY_HPP2 the functionally importantresidues are located in a cleft comprised of residues D65,H94-C95-X-X-G98-X-X-R101 (the ‘active site’). All these residues areconserved in 1vhr (D92, H123-C124-X-X-G127-X-X-R130). Based on thesequence alignment disclosed in FIG. 31 and the structural modeldisclosed in Table IX and visualized in FIG. 32, D65 is identified as ageneral acid, C95 as the catalytic Cysteine nucleophile which cleavesthe phosphodiester bond, and R101 as the essential Argenine whichactivates the bond for cleavage as described in the literature (reviewedby Fauman and Saper, 1996).

In a preferred embodiment of the present invention, the moleculecomprises the cleft region defined by structure coordinates of BMY_HPP2amino acids described above according to Table IX, or a mutant of saidmolecule.

More preferred are molecules comprising all or any part of the cleft ora mutant or homologue of said molecule or molecular complex. By mutantor homologue of the molecule it is meant a molecule that has a root meansquare deviation from the backbone atoms of said BMY_HPP2 amino acids ofnot more than 3.5 Angstroms.

The term “root mean square deviation” means the square root of thearithmetic mean of the squares of the deviations from the mean. It is away to express the deviation or variation from a trend or object. Forpurposes of this invention, the “root mean square deviation” defines thevariation in the backbone of a protein from the relevant portion of thebackbone of BMY_HPP2 as defined by the structure coordinates describedherein.

The structure coordinates of a BMY_HPP2 homology model portions thereofare stored in a machine-readable storage medium. Such data may be usedfor a variety of purposes, such as drug discovery.

Accordingly, in one embodiment of this invention is provided amachine-readable data storage medium comprising a data storage materialencoded with the structure coordinates set forth in Table IX

One embodiment utilizes System 10 as disclosed in WO 98/11134, thedisclosure of which is incorporated herein by reference in its entirety.Briefly, one version of these embodiments comprises a computercomprising a central processing unit (“CPU”), a working memory which maybe, e.g, RAM (random-access memory) or “core” memory, mass storagememory (such as one or more disk drives or CD-ROM drives), one or morecathode-ray tube (“CRT”) display terminals, one or more keyboards, oneor more input lines, and one or more output lines, all of which areinterconnected by a conventional bidirectional system bus.

Input hardware, coupled to the computer by input lines, may beimplemented in a variety of ways. Machine-readable data of thisinvention may be inputted via the use of a modem or modems connected bya telephone line or dedicated data line. Alternatively or additionally,the input hardware may comprise CD-ROM drives or disk drives. Inconjunction with a display terminal, keyboard may also be used as aninput device.

Output hardware, coupled to the computer by output lines, may similarlybe implemented by conventional devices. By way of example, outputhardware may include a CRT display terminal for displaying a graphicalrepresentation of a region or domain of the present invention using aprogram such as QUANTA as described herein. Output hardware might alsoinclude a printer, so that hard copy output may be produced, or a diskdrive, to store system output for later use.

In operation, the CPU coordinates the use of the various input andoutput devices, coordinates data accesses from mass storage, andaccesses to and from the working memory, and determines the sequence ofdata processing steps. A number of programs may be used to process themachine-readable data of this invention. Such programs are discussed inreference to the computational methods of drug discovery as describedherein. Specific references to components of the hardware system areincluded as appropriate throughout the following description of the datastorage medium.

For the purpose of the present invention, any magnetic data storagemedium which can be encoded with machine-readable data would besufficient for carrying out the storage requirements of the system. Themedium could be a conventional floppy diskette or hard disk, having asuitable substrate, which may be conventional, and a suitable coating,which may be conventional, on one or both sides, containing magneticdomains whose polarity or orientation could be altered magnetically, forexample. The medium may also have an opening for receiving the spindleof a disk drive or other data storage device.

The magnetic domains of the coating of a medium may be polarized ororiented so as to encode in a manner which may be conventional, machinereadable data such as that described herein, for execution by a systemsuch as the system described herein.

Another example of a suitable storage medium which could also be encodedwith such machine-readable data, or set of instructions, which could becarried out by a system such as the system described herein, could be anoptically-readable data storage medium. The medium could be aconventional compact disk read only memory (CD-ROM) or a rewritablemedium such as a magneto-optical disk which is optically readable andmagneto-optically writable. The medium preferably has a suitablesubstrate, which may be conventional, and a suitable coating, which maybe conventional, usually of one side of substrate.

In the case of a CD-ROM, as is well known, the coating is reflective andis impressed with a plurality of pits to encode the machine-readabledata. The arrangement of pits is read by reflecting laser light off thesurface of the coating. A protective coating, which preferably issubstantially transparent, is provided on top of the reflective coating.

In the case of a magneto-optical disk, as is well known, the coating hasno pits, but has a plurality of magnetic domains whose polarity ororientation can be changed magnetically when heated above a certaintemperature, as by a laser. The orientation of the domains can be readby measuring the polarization of laser light reflected from the coating.The arrangement of the domains encodes the data as described above.

Thus, in accordance with the present invention, data capable ofdisplaying the three dimensional structure of the BMY_HPP2 homologymodel, or portions thereof and their structurally similar homologues isstored in a machine-readable storage medium, which is capable ofdisplaying a graphical three-dimensional representation of thestructure. Such data may be used for a variety of purposes, such as drugdiscovery.

For the first time, the present invention permits the use, throughhomology modeling based upon the sequence of BMY_HPP2 (FIG. 21; SEQ IDNO:152) of structure-based or rational drug design techniques to design,select, and synthesize chemical entities that are capable of modulatingthe biological function of BMY_HPP2.

Accordingly, the present invention is also directed to the entiresequence in FIG. 21 or any portion thereof for the purpose of generatinga homology model for the purpose of 3D structure-based drug design.

For purposes of this invention, we include mutants or homologues of thesequence in FIG. 21 or any portion thereof. In a preferred embodiment,the mutants or homologues have at least 25% identity, more preferably50% identity, more preferably 75% identity, and most preferably 90%identity to the amino acid residues in FIG. 21.

The three-dimensional model structure of the BMY_HPP2 will also providemethods for identifying modulators of biological function. Variousmethods or combination thereof can be used to identify these compounds.

Structure coordinates of the catalytic region defined above can also beused to identify structural and chemical features. Identified structuralor chemical features can then be employed to design or select compoundsas potential BMY_HPP2 modulators. By structural and chemical features itis meant to include, but is not limited to, van der Waals interactions,hydrogen bonding interactions, charge interaction, hydrophobic bondinginteraction, and dipole interaction. Alternatively, or in conjunction,the three-dimensional structural model can be employed to design orselect compounds as potential BMY_HPP2 modulators. Compounds identifiedas potential BMY_HPP2 modulators can then be synthesized and screened inan assay characterized by binding of a test compound to the BMY_HPP2, orin characterizing BMY_HPP2 deactivation in the presence of a smallmolecule. Examples of assays useful in screening of potential BMY_HPP2modulators include, but are not limited to, screening in silico, invitro assays and high throughput assays. Finally, these methods may alsoinvolve modifying or replacing one or more amino acids from BMY_HPP2according to Table IX.

However, as will be understood by those of skill in the art upon thisdisclosure, other structure based design methods can be used. Variouscomputational structure based design methods have been disclosed in theart.

For example, a number of computer modeling systems are available inwhich the sequence of the BMY_HPP2 and the BMY_HPP2 structure (i.e.,atomic coordinates of BMY_HPP2 and/or the atomic coordinates of theactive site as provided in Table IX) can be input. This computer systemthen generates the structural details of one or more these regions inwhich a potential BMY_HPP2 modulator binds so that complementarystructural details of the potential modulators can be determined. Designin these modeling systems is generally based upon the compound beingcapable of physically and structurally associating with BMY_HPP2. Inaddition, the compound must be able to assume a conformation that allowsit to associate with BMY_HPP2. Some modeling systems estimate thepotential inhibitory or binding effect of a potential BMY_HPP2 modulatorprior to actual synthesis and testing.

Methods for screening chemical entities or fragments for their abilityto associate with a given protein target are also well known. Oftenthese methods begin by visual inspection of the binding site on thecomputer screen. Selected fragments or chemical entities are thenpositioned in one or more of the active site region in BMY_HPP2. Dockingis accomplished using software such as INSIGHTII, QUANTA and SYBYL,following by energy minimization and molecular dynamics with standardmolecular mechanic forcefields such as CHARMM and AMBER. Examples ofcomputer programs which assist in the selection of chemical fragment orchemical entities useful in the present invention include, but are notlimited to, GRID (Goodford, 1985), AUTODOCK (Goodsell, 1990), and DOCK(Kuntz et al. 1982).

Upon selection of preferred chemical entities or fragments, theirrelationship to each other and BMY_HPP2 can be visualized and thenassembled into a single potential modulator. Programs useful inassembling the individual chemical entities include, but are not limitedto SYBYL and LeapFrog (Tripos Associates, St. Louis Mo.), LUDI (Bohm1992) and 3D Database systems (Martin 1992).

Alternatively, compounds may be designed de novo using either an emptyactive site or optionally including some portion of a known inhibitor.Methods of this type of design include, but are not limited to LUDI(Bohm 1992) and LeapFrog (Tripos Associates, St. Louis Mo.).

In addition, BMY_HPP2 is overall well suited to modern methods includingcombinatorial chemistry.

Programs such as DOCK (Kuntz et al. 1982) can be used with the atomiccoordinates from the homology model to identify potential ligands fromdatabases or virtual databases which potentially bind the in the metalbinding region, and which may therefore be suitable candidates forsynthesis and testing.

Additionally, the three-dimensional homology model of BMY_HPP2 will aidin the design of mutants with altered biological activity.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:151 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides consisting of a nucleotide sequence described bythe general formula of a−b, where a is any integer between 1 to 864 ofSEQ ID NO:151, b is an integer between 15 to 878, where both a and bcorrespond to the positions of nucleotide residues shown in SEQ IDNO:151, and where b is greater than or equal to a+14.

Features of the Polypeptide Encoded by Gene No:3

The polypeptide fragment corresponding to this gene provided as SEQ IDNO:8 (FIG. 3), encoded by the polynucleotide sequence according to SEQID NO:7 (FIG. 3), and/or encoded by the polynucleotide contained withinthe deposited clone, BMY_HPP3, has significant homology at thenucleotide and amino acid level to a number of phosphatases, whichinclude, for example, the human protein tyrosine phosphatase PTPCAAX1protein (HS_PTPCAAX1; Genbank Accession No:gi| AAB40597; SEQ ID NO:33);the human protein tyrosine phosphatase PTPCAAX2 (HS_PTPCAAX2; GenbankAccession No:gi| AAB40598; SEQ ID NO:34); the mouse prenylated proteintyrosine phosphatase (MM_PTPCAAX; Genbank Accession No:gi| JC5981; SEQID NO:35); and the Drosophila PRL-1 protein (DM_PRL1; Genbank AccessionNo:gi| AAF53506; SEQ ID NO:36) as determined by BLASTP. An alignment ofthe human phosphatase polypeptide with these proteins is provided inFIG. 8.

BMY_HPP3 is predicted to be a prenylated phosphoprotein phosphatasebased on its similarity to drosophila, mouse and human prenylatedphosphotyrosine phosphatases (PTPCAAX proteins). Among the conservedcatalytic residues, there is a conserved Aspartate (“D”) and a conservednucleophilic Cysteine (“C”) as shown in FIG. 8. At the C-terminus, aconsensus prenylation site is conserved in BMY-HPP3 suggesting that theprotein could be post-translationally modified by farnesylation orgeranylation.

Preferred polynucleotides of the present invention comprise thefollowing nucleic acid sequence:ATGGCTAGAATGAACCTCCCTGCTTCTGTGGACATTGCATACAAAAATGTGAGATTTCTTATTACACACAACCCAACCAATACCTACTTTAATAGATTCTTACAGGAACTTAAGCAGGATGGAGTTACCACCATAGTAAGAGTATGAAAAGCAACTTACAACATTGCTCTTTAGAGAAGGGAAGCATCCAGGTTCCGGACTGGCCTTTGATGATGGTACAGCACCATCCAGCCAGATAATTGATAACTGGTTAAAACTTATGAAAAATAAATTTCATGAAGATCCTGGTTGTTGTATTGCAATTCACTGTGTTGTAGGTTTTGGGTGAGCTCCAGTTGCTAGTTGCCCTAGCTTTAATTGAAGGTGGAATGAAATATGAAAATGTAGTACAGTTCATCAGATAAAAGTGACATGGAACTTTTAACAGCAAACAACTTTTGTATTTGGAGAAATATTGTCTTAAAATATGCTTGCACCTCAGAAATCCCAGAAAT AACTGTTTCCTTCAG(SEQ ID NO: 83). Polypeptides encoding by these polynucleotides are alsoprovided.

Preferred polypeptides of the present invention comprise the followingamino acid sequence:MARMNLPASVDIAYKNVRFLITHNPTNTYFNRFLQELKQDGVTTIVRVKATYNIALLEKGSIQVPDWPFDDGTAPSSQIIDNWLKLMKNKFHEDPGCCIAIHCVVGFGELQLLVALALIEGGMKYENVVQFIRKHGTFNSKQLLYLEKYCLKICLHLRNPRNNCFLQ (SEQ ID NO:84).Polynucleotides encoding these polypeptides are also provided.

Based upon the strong homology to members of the phosphatase proteins,the polypeptide encoded by the human BMY_HPP3 phosphatase of the presentinvention is expected to share at least some biological activity withphosphatase proteins, preferably with members of the novelphosphotyrosine/dual-specificity (P-Tyr, P-Ser and P-Thr) phosphatases,particularly the novel phosphotyrosine/dual-specificity (P-Tyr, P-Serand P-Thr) phosphatases referenced herein.

The present invention encompasses the use of BMY_HPP3 inhibitors and/oractivators of BMY_HPP3 activity for the treatment, detection,amelioaration, or prevention of phosphatase associated disorders,including but not limited to metabolic diseases such as diabetes, inaddition to neural and/or cardiovascular diseases and disorders. Thepresent invention also encompasses the use of BMY_HPP3 inhibitors and/oractivators of BMY_HPP3 activity as immunosuppressive agents,anti-inflammatory agents, and/or anti-tumor agents.

The present invention encompasses the use of BMY_HPP3 phosphataseinhibitors, including, antagonists such as antisense nucleic acids, inaddition to other antagonists, as described herein, in a therapeuticregimen to diagnose, prognose, treat, ameliorate, and/or preventdiseases where a kinase activity is insufficient. One, non-limitingexample of a disease which may occur due to insufficient kinase activityare certain types of diabetes, where one or more kinases involved in theinsulin receptor signal pathway may have insufficient activity orinsufficient expression, for example.

Moreover, the present invention encompasses the use of BMY_HPP3phosphatase activators, and/or the use of the BMY_HPP3 phosphatase geneor protein in a gene therapy regimen, as described herein, for thediagnoses, prognoses, treatment, amelioration, and/or prevention ofdiseases and/or disorders where a kinase activity is overly high, suchas a cancer where a kinase oncogene product has excessive activity orexcessive expression.

The present invention also encompasses the use of catalytically inactivevariants of BMY_HPP3 proteins, including fragments thereof, such as aprotein therapeutic, or the use of the encoding polynucleotide sequenceor as gene therapy, for example, in the diagnoses, prognosis, treatment,amelioration, and/or prevention of diseases or disorders wherephosphatase activity is overly high.

The present invention encompasses the use of antibodies directed againstthe BMY_HPP3 polypeptides, including fragment and/or variants thereof,of the present invention in diagnostics, as a biomarkers, and/or as atherapeutic agents.

The present invention encompasses the use of an inactive, non-catalytic,mutant of the BMY_HPP3 phosphatase as a substrate trapping mutant tobind cellular phosphoproteins or a library of phosphopeptides toidentify substrates of the BMY_HPP3 polypeptides.

The present invention encompasses the use of the BMY_HPP3 polypeptides,to identify inhibitors or activators of the BMY_HPP3 phosphataseactivity using either in vitro or ‘virtual’ (in silico) screeningmethods.

One embodiment of the invention relates to a method for identifying acompound as an activator or inhibitor of the BMY_HPP3 phosphatasecomprising the steps of: i.) contacting a BMY_HPP3 phosphatase inhibitoror activator labeled with an analytically detectable reagent with theBMY_HPP3 phosphatase under conditions sufficient to form a complex withthe inhibitor or activator; ii.) contacting said complex with a samplecontaining a compound to be identified; iii) and identifying thecompound as an inhibitor or activator by detecting the ability of thetest compound to alter the amount of labeled known BMY_HPP3 phosphataseinhibitor or activator in the complex.

Another embodiment of the invention relates to a method for identifyinga compound as an activator or inhibitor of a BMY_HPP3 phosphatasecomprising the steps of: i.) contacting the BMY_HPP3 phosphatase with acompound to be identified; and ii.) and measuring the ability of theBMY_HPP3 phosphatase to remove phosphate from a substrate.

The present invention also encomposses a method for identifying a ligandfor the BMY_HPP3 phosphatase comprising the steps of: i.) contacting theBMY_HPP3 phosphatase with a series of compounds under conditions topermit binding; and ii.) detecting the presence of any ligand-boundprotein.

Preferably, the above referenced methods comprise the BMY_HPP3phosphatase in a form selected from the group consisting of whole cells,cytosolic cell fractions, membrane cell fractions, purified or partiallypurified forms. The invention also relates to recombinantly expressedBMY_HPP3 phosphatase in a purified, substantially purified, orunpurified state. The invention further relates to BMY_HPP3 phosphatasefused or conjugated to a protein, peptide, or other molecule or compoundknown in the art, or referenced herein.

The present invention also encompasses pharmaceutical composition of theBMY_HPP3 phosphatase polypeptide comprising a compound identified byabove referenced methods and a pharmaceutically acceptable carrier.

Features of the Polypeptide Encoded by Gene No:4

The polypeptide fragment corresponding to this gene provided as SEQ IDNO:10 (FIG. 4), encoded by the polynucleotide sequence according to SEQID NO:9 (FIG. 4), and/or encoded by the polynucleotide contained withinthe deposited clone, BMY_HPP4, has significant homology at thenucleotide and amino acid level to a number of phosphatases, whichinclude, for example, the mouse osteotesticular protein tyrosinephosphatase (MM_OST-PTP; Genbank Accession No:gi| AAG28768; SEQ IDNO:37); and the rat protein-tyrosine-phosphatase (RN_PTP-OST; GenbankAccession No:gi| A55148; SEQ ID NO:38) as determined by BLASTP. Analignment of the human phosphatase polypeptide with these proteins isprovided in FIG. 9.

BMY_HPP4 is predicted to be a phosphoprotein phosphatase based on itshomology to rat osteotesticular receptor protein-tyrosine-phosphataseprecursor (Genbank ID 1083770) and to mouse receptorprotein-tyrosine-phosphatase precursor (Genbank ID 11066925). TheBMY_HPP4 polypeptide has been shown to comprise a conserved Aspartate(“D”) at amino acid 182 of SEQ ID NO:10 (FIG. 4), a catalytic Cysteine(“C”) at amino acid 216 of SEQ ID NO:10 (FIG. 4), and a conservedArgenine (“R”) at amino acid 227 of SEQ ID NO:10 (FIG. 4).

The predicted exon structure of the BMY_HPP4 gene is provided in TableV. The ‘Start’ and ‘End’ designations refer to the respective nucleotidepositions of the BMY_HPP4 as they appear for BAC AL 354751. Thenumbering begins at the start of BAC AL354751; nucleotide 71352 in theBAC is equivalent to nucleotide 1 of the BMY_HPP4 transcript (SEQ IDNO:9; FIG. 4).

Based upon the strong homology to members of the phosphatase proteins,the polypeptide encoded by the human BMY_HPP4 phosphatase of the presentinvention is expected to share at least some biological activity withphosphatase proteins, preferably with members of the novelphosphotyrosine/dual-specificity (P-Tyr, P-Ser and P-Thr) phosphatases,particularly the novel phosphotyrosine/dual-specificity (P-Tyr, P-Serand P-Thr) phosphatases referenced herein.

The present invention encompasses the use of BMY_HPP4 inhibitors and/oractivators of BMY_HPP4 activity for the treatment, detection,amelioaration, or prevention of phosphatase associated disorders,including but not limited to metabolic diseases such as diabetes, inaddition to neural and/or cardiovascular diseases and disorders. Thepresent invention also encompasses the use of BMY_HPP4 inhibitors and/oractivators of BMY_HPP4 activity as immunosuppressive agents,anti-inflammatory agents, and/or anti-tumor agents.

The present invention encompasses the use of BMY_HPP4 phosphataseinhibitors, including, antagonists such as antisense nucleic acids, inaddition to other antagonists, as described herein, in a therapeuticregimen to diagnose, prognose, treat, ameliorate, and/or preventdiseases where a kinase activity is insufficient. One, non-limitingexample of a disease which may occur due to insufficient kinase activityare certain types of diabetes, where one or more kinases involved in theinsulin receptor signal pathway may have insufficient activity orinsufficient expression, for example.

Moreover, the present invention encompasses the use of BMY_HPP4phosphatase activators, and/or the use of the BMY_HPP4 phosphatase geneor protein in a gene therapy regimen, as described herein, for thediagnoses, prognoses, treatment, amelioration, and/or prevention ofdiseases and/or disorders where a kinase activity is overly high, suchas a cancer where a kinase oncogene product has excessive activity orexcessive expression.

The present invention also encompasses the use of catalytically inactivevariants of BMY_HPP4 proteins, including fragments thereof, such as aprotein therapeutic, or the use of the encoding polynucleotide sequenceor as gene therapy, for example, in the diagnoses, prognosis, treatment,amelioration, and/or prevention of diseases or disorders wherephosphatase activity is overly high.

The present invention encompasses the use of antibodies directed againstthe BMY_HPP4 polypeptides, including fragment and/or variants thereof,of the present invention in diagnostics, as a biomarkers, and/or as atherapeutic agents.

The present invention encompasses the use of an inactive, non-catalytic,mutant of the BMY_HPP4 phosphatase as a substrate trapping mutant tobind cellular phosphoproteins or a library of phosphopeptides toidentify substrates of the BMY_HPP4 polypeptides.

The present invention encompasses the use of the BMY_HPP4 polypeptides,to identify inhibitors or activators of the BMY_HPP4 phosphataseactivity using either in vitro or ‘virtual’ (in silico) screeningmethods.

One embodiment of the invention relates to a method for identifying acompound as an activator or inhibitor of the BMY_HPP4 phosphatasecomprising the steps of: i.) contacting a BMY_HPP4 phosphatase inhibitoror activator labeled with an analytically detectable reagent with theBMY_HPP4 phosphatase under conditions sufficient to form a complex withthe inhibitor or activator; ii.) contacting said complex with a samplecontaining a compound to be identified; iii) and identifying thecompound as an inhibitor or activator by detecting the ability of thetest compound to alter the amount of labeled known BMY_HPP4 phosphataseinhibitor or activator in the complex.

Another embodiment of the invention relates to a method for identifyinga compound as an activator or inhibitor of a BMY_HPP4 phosphatasecomprising the steps of: i.) contacting the BMY_HPP4 phosphatase with acompound to be identified; and ii.) and measuring the ability of theBMY_HPP4 phosphatase to remove phosphate from a substrate.

The present invention also encomposses a method for identifying a ligandfor the BMY_HPP4 phosphatase comprising the steps of: i.) contacting theBMY_HPP4 phosphatase with a series of compounds under conditions topermit binding; and ii.) detecting the presence of any ligand-boundprotein.

Preferably, the above referenced methods comprise the BMY_HPP4phosphatase in a form selected from the group consisting of whole cells,cytosolic cell fractions, membrane cell fractions, purified or partiallypurified forms. The invention also relates to recombinantly expressedBMY_HPP4 phosphatase in a purified, substantially purified, orunpurified state. The invention further relates to BMY_HPP4 phosphatasefused or conjugated to a protein, peptide, or other molecule or compoundknown in the art, or referenced herein.

The present invention also encompasses pharmaceutical composition of theBMY_HPP4 phosphatase polypeptide comprising a compound identified byabove referenced methods and a pharmaceutically acceptable carrier.

Expression profiling of the BMY_HPP4 polypeptide in normal tissuesshowed that BMY_HPP4 is expressed at higher levels in the cerebellumthan in any other tissue, suggesting a role for modulators of BMY_HPP4activity in the treatment of neurological disorders such as depression,bipolar disorder, schizophrenia, dementia and cognitive disorders (asshown in FIG. 34). BMY_HPP4 was also expressed at lower levels in othersubregions of the brain. In addition, BMY_HPP4 was expressed atsignificant levels in the pineal and pituitary glands, suggesting a rolefor modulators of BMY_HPP4 activity in endocrine disorders.

The strong homology to dual specificity phophatases, combined with thelocalized expression in cerebellum suggests the BMY_HPP4 polynucleotidesand polypeptides may be useful in treating, diagnosing, prognosing,and/or preventing neurodegenerative disease states, behavioraldisorders, or inflammatory conditions. Representative uses are describedin the “Regeneration” and “Hyperproliferative Disorders” sections below,in the Examples, and elsewhere herein. Briefly, the uses include, butare not limited to the detection, treatment, and/or prevention ofAlzheimer's Disease, Parkinson's Disease, Huntington's Disease, TouretteSyndrome, meningitis, encephalitis, demyelinating diseases, peripheralneuropathies, neoplasia, trauma, congenital malformations, spinal cordinjuries, ischemia and infarction, aneurysms, hemorrhages,schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder,depression, panic disorder, learning disabilities, ALS, psychoses,autism, and altered behaviors, including disorders in feeding, sleeppatterns, balance, and perception. In addition, elevated expression ofthis gene product in regions of the brain indicates it plays a role innormal neural function. Potentially, this gene product is involved insynapse formation, neurotransmission, learning, cognition, homeostasis,or neuronal differentiation or survival. Furthermore, the protein mayalso be used to determine biological activity, to raise antibodies, astissue markers, to isolate cognate ligands or receptors, to identifyagents that modulate their interactions, in addition to its use as anutritional supplement. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

The strong homology to dual specificity phophatases, combined with thelocalized expression in pineal and pituitary glands suggests theBMY_HPP4 polynucleotides and polypeptides may be useful in treating,diagnosing, prognosing, and/or preventing endocrine diseases and/ordisorders, which include, but are not limited to, the following:aberrant growth hormone synthesis and/or secretion, aberrant prolactinsynthesis and/or secretion, aberrant luteinizing hormone synthesisand/or secretion, aberrant follicle-stimulating hormone synthesis and/orsecretion, aberrant thyroid-stimulating hormone synthesis and/orsecretion, aberrant adrenocorticotropin synthesis and/or secretion,aberrant vasopressin secretion, aberrant oxytocin secretion, aberrantgrowth, aberrant lactation, aberrant sexual characteristic development,aberrant testosterone synthesis and/or secretion, aberrant estrogensynthesis and/or secretion, aberrant water homeostasis, hypogonadism,Addison's disease, hypothyroidism, Cushing's disease, agromegaly,gigantism, lethargy, osteoporosis, aberrant calcium homeostasis,aberrant potassium homeostasis, reproductive disorders, anddevelopmental disorders.

Features of the Polypeptide Encoded by Gene No:5

The polypeptide corresponding to this gene provided as SEQ ID NO:42(FIG. 5), encoded by the polynucleotide sequence according to SEQ IDNO:41 (FIG. 5), and/or encoded by the polynucleotide contained withinthe deposited clone, BMY_HPP5, has significant homology at thenucleotide and amino acid level to a number of phosphatases, whichinclude, for example, the human dual specificity phosphatase 8(hs_dspp8; Genbank Accession No:gi| NP_(—)004411; SEQ ID NO:39); and themouse neuronal tyrosine/threonine phosphatase 1 (r mm_npp1; GenbankAccession No:gi| NP_(—)032774; SEQ ID NO:40) as determined by BLASTP. Analignment of the human phosphatase polypeptide with these proteins isprovided in FIGS. 10A-B.

The determined nucleotide sequence of the BMY_HPP5 cDNA in FIGS. 5A-E(SEQ ID NO:41) contains an open reading frame encoding a protein ofabout 665 amino acid residues, with a deduced molecular weight of about73 kDa. The amino acid sequence of the predicted BMY_HPP5 polypeptide isshown in FIGS. 5A-E (SEQ ID NO:42). The BMY_HPP5 protein shown in FIGS.5A-E was determined to share significant identity and similarity toseveral known phosphatases, particularly, dual-specificity proteinphosphatases. Specifically, the BMY_HPP5 protein shown in FIGS. 5A-E wasdetermined to be about 46% identical and 58% similar to the human dualspecificity phosphatase 8 (HS_DSPP8; Genbank Accession No: gi|NP_(—)004411; SEQ ID NO:39); and about 43% identical and 56% similar tothe mouse neuronal tyrosine/threonine phosphatase 1 (MM_NPP1; GenbankAccession No: gi| NP_(—)032774; SEQ ID NO:40), as shown in FIG. 12.

BMY_HPP5 is predicted to encode a phosphoprotein phosphatase based onits homology to known dual-specificity protein phosphatases includinghuman dual-specificity protein phosphatase 8 (GI 4758212) and mouseneuronal tyrosine/threonine phosphatase I (GI 6679156) (FIGS. 10A-B).The BMY_HPP5 polypeptide was determined to comprise conserved residues,which include, the catalytic Aspartate (“D”) at amino acid 212, and aconserved Cysteine (“C”) at amino acid 244, and Arginine (“R”) at aminoacid 249 of SEQ ID NO:42 (FIGS. 5A-E).

Based upon the strong homology to members of the phosphatase proteins,the polypeptide encoded by the human BMY_HPP5 phosphatase of the presentinvention is expected to share at least some biological activity withphosphatase proteins, preferably with members of the novelphosphotyrosine/dual-specificity (P-Tyr, P-Ser and P-Thr) phosphatases,particularly the novel phosphotyrosine/dual-specificity (P-Tyr, P-Serand P-Thr) phosphatases referenced herein.

Expression profiling designed to measure the steady state mRNA levelsencoding the human phosphatase polypeptide, BMY_HPP5, showedpredominately high expression levels in the testis and spinal cord, andto a lesser extent, in bone marrow, brain, liver, and thymus. (See FIG.11).

Moreover, expanded expression profiling of the BMY_HPP5 polypeptide innormal human tissues showed the highest levels of expression in theadrenal, pineal and pituitary glands suggesting that modulators ofBMY_HPP5 activity could be useful in the treatment of endocrinedisorders (as shown in FIG. 35). BMY_HPP5 also expressed at high levelsin the cerebellum, suggesting a role for modulators of BMY_HPP5 activityin the treatment of neurological disorders such as depression, bipolardisorder, schizophrenia, dementia and cognitive disorders; in theprostate, suggesting a role for modulators of BMY_HPP5 activity in thetreatment of prostate cancer or benign prostatic hyperplasia; in thetestis, suggesting a role for modulators of BMY_HPP5 activity in thetreatment of male infertility caused by defective or insufficientspermatogenesis, as a contraceptive agent, or in the treatment oftesticular cancer. BMYBMY_HPP5 was also expressed at a lower butsignificant level in many other normal human tissues.

The strong homology to phosphatases, particularly dual-specificityphosphatases, combined with the predominate localized expression inadrenal gland tissue suggests the human BMY_HPP5 phosphatasepolynucleotides and polypeptides, including antagonists, and/orfragments thereof, may be useful for treating, diagnosing, prognosing,ameliorating, and/or preventing endocrine disorders, which include, butare mot limited to adrenocortical hyperfunction, adrenocorticalhypofunction, lethargy. Congenital adrenal hyperplasia, aberrant ACTHregulation, aberrant adrenaline regulation, disorders associated withdefects in P450C21, P450C18, P450C17, and P450C11 hydroxylases and in3-hydroxysteroid dehydrogenase (3-HSD), hirsutism, oligomenorrhea, acne,virilization, oligomenorrhea, female pseudohermaphroditism, disordersassociated with the incidence of aberrant sexual characterisitics,disorders associated with aberrant cortisol secretion, hypertension,hypokalemia, hypogonadism, disorders associated with aberrant androgensecretion, adrenal virilism, Adrenal adenomas, Adrenal carcinomas,disorders associated with aberrant aldosterone secretion, aldosteronism,disorders associated with aberrant steriod biosynthesis, disordersassociated with aberrant steriod transport, disorders associated withaberrant steriod secretion, disorders associated with aberrant steriodexcretion, Addison's syndrome, and Cushing's syndrome.

The strong homology to phosphatases, particularly dual-specificityphosphatases, combined with the predominate localized expression inpituitary gland tissue suggests the BMY_HPP5 polynucleotides andpolypeptides may be useful in treating, diagnosing, prognosing, and/orpreventing endocrine diseases and/or disorders, which include, but arenot limited to, the following: aberrant growth hormone synthesis and/orsecretion, aberrant prolactin synthesis and/or secretion, aberrantluteinizing hormone synthesis and/or secretion, aberrantfollicle-stimulating hormone synthesis and/or secretion, aberrantthyroid-stimulating hormone synthesis and/or secretion, aberrantadrenocorticotropin synthesis and/or secretion, aberrant vasopressinsecretion, aberrant oxytocin secretion, aberrant growth, aberrantlactation, aberrant sexual characteristic development, aberranttestosterone synthesis and/or secretion, aberrant estrogen synthesisand/or secretion, aberrant water homeostasis, hypogonadism, Addison'sdisease, hypothyroidism, Cushing's disease, agromegaly, gigantism,lethargy, osteoporosis, aberrant calcium homeostasis, aberrant potassiumhomeostasis, reproductive disorders, developmental disorders, anddepression related to low incident light levels.

The strong homology to phosphatases, particularly dual-specificityphosphatases, combined with the predominate localized expression intestis tissue suggests the human BMY_HPP5 phosphatase polynucleotidesand polypeptides, including antagonists, and/or fragments thereof, maybe useful for treating, diagnosing, prognosing, and/or preventing malereproductive disorders, such as, for example, male infertility,impotence, and/or testicular cancer. This gene product may also beuseful in assays designed to identify binding agents, as such agents(antagonists) are useful as male contraceptive agents. The testes arealso a site of active gene expression of transcripts that is expressed,particularly at low levels, in other tissues of the body. Therefore,this gene product may be expressed in other specific tissues or organswhere it may play related functional roles in other processes, such ashematopoiesis, inflammation, bone formation, and kidney function, toname a few possible target indications. If fact, increased expression ofcertain phosphatases have been identified as tumor markers fortesticular cancer (see, for example, Koshida, K., Nishino, A., Yamamoto,H., Uchibayashi, T., Naito, K., Hisazumi, H., Hirano, K., Hayashi, Y.,Wahren, B., Andersson, L, J. Urol., 146(1):57-60, (1991); and Klein, EA, Urol. Clin. North. Am., 20(1):67-73, (1993)).

Alternatively, the strong homology to phosphatases, particularlydual-specificity phosphatases, combined with the significant localizedexpression in spinal cord and brain tissue suggests the humanphosphatase polynucleotides and polypeptides may be useful in treating,diagnosing, prognosing, and/or preventing neural diseases and/ordisorders. Representative uses are described in the “NeurologicalDiseases” section below, and elsewhere herein. Briefly, the expressionin neural tissue indicates a role in Alzheimer's Disease, Parkinson'sDisease, Huntington's Disease, Tourette Syndrome, meningitis,encephalitis, demyelinating diseases, peripheral neuropathies,neoplasia, trauma, congenital malformations, spinal dyphida, spinal cordinjuries, ischemia and infarction, aneurysms, hemorrhages,schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder,depression, panic disorder, learning disabilities, ALS, psychoses,autism, and altered behaviors, including disorders in feeding, sleeppatterns, balance, and perception. In addition, elevated expression ofthis gene product in regions of the brain indicates it plays a role innormal neural function. Potentially, this gene product is involved insynapse formation, neurotransmission, learning, cognition, homeostasis,or neuronal differentiation or survival. Furthermore, the protein mayalso be used to determine biological activity, to raise antibodies, astissue markers, to isolate cognate ligands or receptors, to identifyagents that modulate their interactions, in addition to its use as anutritional supplement. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

Moreover, the tissue distribution in liver indicates the protein productof this clone would be useful for the detection and treatment of liverdisorders and cancers. Representative uses are described in the“Hyperproliferative Disorders”, “Infectious Disease”, and “BindingActivity” sections below, and elsewhere herein. Briefly, the protein canbe used for the detection, treatment, and/or prevention ofhepatoblastoma, jaundice, hepatitis, liver metabolic diseases andconditions that are attributable to the differentiation of hepatocyteprogenitor cells. In addition the expression in fetus would suggest auseful role for the protein product in developmental abnormalities,fetal deficiencies, pre-natal disorders and various would-healingdiseases and/or tissue trauma.

Moreover, human phosphatase polynucleotides and polypeptides, includingfragments and agonists thereof, may have uses which include treating,diagnosing, prognosing, and/or preventing hyperproliferative disorders,particularly of the renal, neural, and reproductive systems. Suchdisorders may include, for example, cancers, and metastasis.

The human phosphatase polynucleotides and polypeptides, includingfragments and agonists thereof, may have uses which include, eitherdirectly or indirectly, for boosting immune responses.

The human phosphatase polynucleotides and polypeptides, includingfragments and/or antagonists thereof, may have uses which includeidentification of modulators of human phosphatase function includingantibodies (for detection or neutralization), naturally-occurringmodulators and small molecule modulators. Antibodies to domains of thehuman phosphatase protein could be used as diagnostic agents ofcardiovascular and inflammatory conditions in patients, are useful inmonitoring the activation of signal transduction pathways, and can beused as a biomarker for the involvement of phosphatases in diseasestates, and in the evaluation of inhibitors of phosphatases in vivo.

Human phosphatase polypeptides and polynucleotides have additional useswhich include diagnosing diseases related to the over and/or underexpression of human phosphatase by identifying mutations in the humanphosphatase gene by using human phosphatase sequences as probes or bydetermining human phosphatase protein or mRNA expression levels. Humanphosphatase polypeptides may be useful for screening compounds thataffect the activity of the protein. Human phosphatase peptides can alsobe used for the generation of specific antibodies and as bait in yeasttwo hybrid screens to find proteins the specifically interact with humanphosphatase (described elsewhere herein).

Although it is believed the encoded polypeptide may share at least somebiological activities with phosphatase proteins (particularly dualspecificity proteins), a number of methods of determining the exactbiological function of this clone are either known in the art or aredescribed elsewhere herein. Briefly, the function of this clone may bedetermined by applying microarray methodology. Nucleic acidscorresponding to the human phosphatase polynucleotides, in addition to,other clones of the present invention, may be arrayed on microchips forexpression profiling. Depending on which polynucleotide probe is used tohybridize to the slides, a change in expression of a specific gene mayprovide additional insight into the function of this gene based upon theconditions being studied. For example, an observed increase or decreasein expression levels when the polynucleotide probe used comes fromdiseased heart tissue, as compared to, normal tissue might indicate afunction in modulating cardiac function, for example. In the case ofhuman BMY_HPP5 phosphatase, testis, spinal cord, brain, liver, bonemarrow, and thymus tissue should be used, for example, to extract RNA toprepare the probe.

In addition, the function of the protein may be assessed by applyingquantitative PCR methodology, for example. Real time quantitative PCRwould provide the capability of following the expression of the humanphosphatase gene throughout development, for example. Quantitative PCRmethodology requires only a nominal amount of tissue from eachdevelopmentally important step is needed to perform such experiments.Therefore, the application of quantitative PCR methodology to refiningthe biological function of this polypeptide is encompassed by thepresent invention. In the case of human phosphatase, a diseasecorrelation related to human phosphatase may be made by comparing themRNA expression level of human phosphatase in normal tissue, as comparedto diseased tissue (particularly diseased tissue isolated from thefollowing: testis, spinal cord, brain, liver, bone marrow, and thymustissue). Significantly higher or lower levels of human phosphataseexpression in the diseased tissue may suggest human phosphatase plays arole in disease progression, and antagonists against human phosphatasepolypeptides would be useful therapeutically in treating, preventing,and/or ameliorating the disease. Alternatively, significantly higher orlower levels of human phosphatase expression in the diseased tissue maysuggest human phosphatase plays a defensive role against diseaseprogression, and agonists of human phosphatase polypeptides may beuseful therapeutically in treating, preventing, and/or ameliorating thedisease. Also encompassed by the present invention are quantitative PCRprobes corresponding to the polynucleotide sequence provided as SEQ IDNO:41 (FIGS. 4A-D).

The function of the protein may also be assessed through complementationassays in yeast. For example, in the case of the human phosphatase,transforming yeast deficient in purinergic receptor activity, forexample, and assessing their ability to grow would provide convincingevidence the human phosphatase polypeptide has purinergic receptoractivity. Additional assay conditions and methods that may be used inassessing the function of the polynucleotides and polypeptides of thepresent invention are known in the art, some of which are disclosedelsewhere herein.

Alternatively, the biological function of the encoded polypeptide may bedetermined by disrupting a homologue of this polypeptide in Mice and/orrats and observing the resulting phenotype. Such knock-out experimentsare known in the art, some of which are disclosed elsewhere herein.

Moreover, the biological function of this polypeptide may be determinedby the application of antisense and/or sense methodology and theresulting generation of transgenic mice and/or rats. Expressing aparticular gene in either sense or antisense orientation in a transgenicmouse or rat could lead to respectively higher or lower expressionlevels of that particular gene. Altering the endogenous expressionlevels of a gene can lead to the observation of a particular phenotypethat can then be used to derive indications on the function of the gene.The gene can be either over-expressed or under expressed in every cellof the organism at all times using a strong ubiquitous promoter, or itcould be expressed in one or more discrete parts of the organism using awell characterized tissue-specific promoter (e.g., a kidney, lung,spinal cord, or testes tissue specific promoter), or it can be expressedat a specified time of development using an inducible and/or adevelopmentally regulated promoter.

In the case of human phosphatase transgenic mice or rats, if nophenotype is apparent in normal growth conditions, observing theorganism under diseased conditions (renal, pulmonary, neurological, orreproductive disorders, in addition to cancers, etc.) may lead tounderstanding the function of the gene. Therefore, the application ofantisense and/or sense methodology to the creation of transgenic mice orrats to refine the biological function of the polypeptide is encompassedby the present invention.

In preferred embodiments, the following N-terminal deletion mutants areencompassed by the present invention: M1-S665, A2-S665, H3-S665,E4-S665, M5-S665, -S665, G7-S665, T8-S665, Q9-S665, I10-S665, V11-S665,T12-S665, E13-S665, R14-S665, L15-S665, V16-S665, A17-S665, L18-S665,L19-S665, E20-S665, S21-S665, G22-S665, T23-S665, E24-S665, K25-S665,V26-S665, L27-S665, L28-S665, I29-S665, D30-S665, S31-S665, R32-S665,P33-S665, F34-S665, V35-S665, E36-S665, Y37-S665, N38-S665, T39-S665,S40-S665, H41-S665, I42-S665, L43-S665, E44-S665, A45-S665, I46-S665,N47-S665, I48-S665, N49-S665, C50-S665, S51-S665, K52-S665, L53-S665,M54-S665, K55-S665, R56-S665, R57-S665, L58-S665, Q59-S665, Q60-S665,D6-S665, K62-S665, V63-S665, L64-S665, I65-S665, T66-S665, E67-S665,L68-S665, I69-S665, Q70-S665, H71-S665, S72-S665, A73-S665, K74-S665,H75-S665, K76-S665, V77-S665, D78-S665, I79-S665, D80-S665, C81-S665,S82-S665, Q83-S665, K84-S665, V85-S665, V86-S665, V87-S665, Y88-S665,D89-S665, Q90-S665, S91-S665, S92-S665, Q93-S665, D94-S665, V95-S665,A96-S665, S97-S665, L98-S665, S99-S665, S100-S665, D101-S665, C102-S665,F103-S665, L104-S665, T105-S665, V106-S665, L107-S665, L108-S665,G109-S665, K110-S665, L111-S665, E112-S665, K113-S665, S114-S665,F115-S665, N116-S665, S117-S665, V118-S665, H119-S665, L120-S665,L121-S665, A122-S665, G123-S665, G124-S665, F125-S665, A126-S665,E127-S665, F128-S665, S129-S665, R130-S665, C131-S665, F132-S665,P133-S665, G134-S665, L135-S665, C136-S665, E137-S665, G138-S665,K139-S665, S140-S665, T141-S665, L142-S665, V143-S665, P144-S665,T145-S665, C146-S665, I147-S665, S148-S665, Q149-S665, P150-S665,C151-S665, L152-S665, P153-S665, V154-S665, A155-S665, N156-S665,I157-S665, G158-S665, P159-S665, T160-S665, R161-S665, I162-S665,L163-S665, P164-S665, N165-S665, L166-S665, Y167-S665, L168-S665,G169-S665, C170-S665, Q171-S665, R172-S665, D173-S665, V174-S665,L175-S665, N176-S665, K177-S665, E178-S665, L179-S665, M180-S665,Q181-S665, Q182-S665, N183-S665, G184-S665, I185-S665, G186-S665,Y187-S665, V188-S665, L189-S665, N190-S665, A191-S665, S192-S665,N193-S665, T194-S665, C195-S665, P196-S665, K197-S665, P198-S665,D199-S665, F200-S665, I201-S665, P202-S665, E203-S665, S204-S665,H205-S665, F206-S665, L207-S665, R208-S665, V209-S665, P210-S665,V211-S665, N212-S665, D213-S665, S214-S665, F215-S665, C216-S665,E217-S665, K218-S665, I219-S665, L220-S665, P221-S665, W222-S665,L223-S665, D224-S665, K225-S665, S226-S665, V227-S665, D228-S665,F229-S665, I230-S665, E231-S665, K232-S665, A233-S665, K234-S665,A235-S665, S236-S665, N237-S665, G238-S665, C239-S665, V240-S665,L241-S665, V242-S665, H243-S665, C244-S665, L245-S665, A246-S665,G247-S665, I248-S665, S249-S665, R250-S665, S251-S665, A252-S665,T253-S665, I254-S665, A255-S665, I256-S665, A257-S665, Y258-S665,I259-S665, M260-S665, K261-S665, R262-S665, M263-S665, D264-S665,M265-S665, S266-S665, L267-S665, D268-S665, E269-S665, A270-S665,Y271-S665, R272-S665, F273-S665, V274-S665, K275-S665, E276-S665,K277-S665, R278-S665, P279-S665, T280-S665, I281-S665, S282-S665,P283-S665, N284-S665, F285-S665, N286-S665, F287-S665, L288-S665,G289-S665, Q290-S665, L291-S665, L292-S665, A293-S665, Y294-S665,E295-S665, K296-S665, K297-S665, I298-S665, K299-S665, N300-S665,Q301-S665, T302-S665, G303-S665, A304-S665, S305-S665, G306-S665,P307-S665, K308-S665, S309-S665, K310-S665, L311-S665, K312-S665,L313-S665, L314-S665, P315-S665, L316-S665, E317-S665, K318-S665,P319-S665, N320-S665, E321-S665, P322-S665, V323-S665, P324-S665,A325-S665, V326-S665, S327-S665, E328-S665, G329-S665, G330-S665,Q331-S665, K332-S665, S333-S665, E334-S665, T335-S665, P336-S665,L337-S665, S338-S665, P339-S665, P340-S665, C341-S665, A342-S665,D343-S665, S344-S665, A345-S665, T346-S665, S347-S665, E348-S665,A349-S665, A350-S665, G351-S665, Q352-S665, R353-S665, P354-S665,V355-S665, H356-S665, P357-S665, A358-S665, S359-S665, V360-S665,P361-S665, S362-S665, V363-S665, P364-S665, S365-S665, V366-S665,Q367-S665, P368-S665, S369-S665, L370-S665, L371-S665, E372-S665,D373-S665, S374-S665, P375-S665, L376-S665, V377-S665, Q378-S665,A379-S665, L380-S665, S381-S665, G382-S665, L383-S665, H384-S665,L385-S665, S386-S665, A387-S665, D388-S665, R389-S665, L390-S665,E391-S665, D392-S665, S393-S665, N394-S665, K395-S665, L396-S665,K397-S665, R398-S665, S399-S665, F400-S665, S401-S665, L402-S665,D403-S665, I404-S665, K405-S665, S406-S665, V407-S665, S408-S665,Y409-S665, S410-S665, A411-S665, S412-S665, M413-S665, A414-S665,A415-S665, S416-S665, L417-S665, H418-S665, G419-S665, F420-S665,S421-S665, S422-S665, S423-S665, E424-S665, D425-S665, A426-S665,L427-S665, E428-S665, Y429-S665, Y430-S665, K431-S665, P432-S665,S433-S665, T434-S665, T435-S665, L436-S665, D437-S665, G438-S665,T439-S665, N440-S665, K441-S665, L442-S665, C443-S665, Q444-S665,F445-S665, S446-S665, P447-S665, V448-S665, Q449-S665, E450-S665,L451-S665, S452-S665, E453-S665, Q454-S665, T455-S665, P456-S665,E457-S665, T458-S665, S459-S665, P460-S665, D461-S665, K462-S665,E463-S665, E464-S665, A465-S665, S466-S665, I467-S665, P468-S665,K469-S665, K470-S665, L471-S665, Q472-S665, T473-S665, A474-S665,R475-S665, P476-S665, S477-S665, D478-S665, S479-S665, Q480-S665,S481-S665, K482-S665, R483-S665, L484-S665, H485-S665, S486-S665,V487-S665, R488-S665, T489-S665, S490-S665, S491-S665, S492-S665,G493-S665, T494-S665, A495-S665, Q496-S665, R497-S665, S498-S665,S499-S665, L500-S665, S501-S665, P502-S665, L503-S665, H504-S665,R505-S665, S506-S665, G507-S665, S508-S665, V509-S665, E510-S665,D511-S665, N512-S665, Y513-S665, H514-S665, T515-S665, S516-S665,F517-S665, L518-S665, F519-S665, G520-S665, L521-S665, S522-S665,T523-S665, S524-S665, Q525-S665, Q526-S665, H527-S665, L528-S665,T529-S665, K530-S665, S531-S665, A532-S665, G533-S665, L534-S665,G535-S665, L536-S665, K537-S665, G538-S665, W539-S665, H540-S665,S541-S665, D542-S665, I543-S665, L544-S665, A545-S665, P546-S665,Q547-S665, T548-S665, S549-S665, T550-S665, P551-S665, S552-S665,L553-S665, T554-S665, S555-S665, S556-S665, W557-S665, Y558-S665,F559-S665, A560-S665, T561-S665, E562-S665, S563-S665, S564-S665,H565-S665, F566-S665, Y567-S665, S568-S665, A569-S665, S570-S665,A571-S665, I572-S665, Y573-S665, G574-S665, G575-S665, S576-S665,A577-S665, S578-S665, Y579-S665, S580-S665, A581-S665, Y582-S665,S583-S665, C584-S665, S585-S665, Q586-S665, L587-S665, P588-S665,T589-S665, C590-S665, G591-S665, D592-S665, Q593-S665, V594-S665,Y595-S665, S596-S665, V597-S665, R598-S665, R599-S665, R600-S665,Q601-S665, K602-S665, P603-S665, S604-S665, D605-S665, R606-S665,A607-S665, D608-S665, S609-S665, R610-S665, R611-S665, S612-S665,W613-S665, H614-S665, E615-S665, E616-S665, S617S665, P618-S665,F619-S665, E620-S665, K621-S665, Q622-S665, F623-S665, K624-S665,R625-S665, R626-S665, S627-S665, C628-S665, Q629-S665, M630-S665,E631-S665, F632-S665, G633-S665, E634-S665, S635-S665, I636-S665,M637-S665, S638-S665, E639-S665, N640-S665, R641-S665, S642-S665,R643-S665, E644-S665, E645-S665, L646-S665, G647-S665, K648-S665,V649-S665, G650-S665, S651-S665, Q652-S665, S653-S665, S654-S665,F655-S665, S656-S665, G657-S665, S658-S665, and/or M659-S665 of SEQ IDNO:42. Polynucleotide sequences encoding these polypeptides are alsoprovided. The present invention also encompasses the use of the humanBMY_HPP5 phosphatase N-terminal deletion polypeptides as immunogenicand/or antigenic epitopes as described elsewhere herein.

In preferred embodiments, the following C-terminal deletion mutants areencompassed by the present invention: M1-S665, M1-V664, M1-E663,M1-I662, M1-I661, M1-E660, M1-M659, M1-S658, M1-G657, M1-S656, M1-F655,M1-S654, M1-S653, M1-Q652, M1-S651, M1-G650, M1-V649, M1-K648, M1-G647,M1-L646, M1-E645, M1-E644, M1-R643, M1-S642, M1-R641, M1-N640, M1-E639,M1-S638, M1-M637, M1-I636, M1-S635, M1-E634, M1-G633, M1-F632, M1-E631,M1-M630, M1-Q629, M1-C628, M1-S627, M1-R626, M1-R625, M1-K624, M1-F623,M1-Q622, M1-K621, M1-E620, M1-F619, M1-P618, M1-S617, M1-E616, M1-E615,M1-H614, M1-W613, M1-S612, M1-R611, M1-R610, M1-S609, M1-D608, M1-A607,M1-R606, M1-D605, M1-S604, M1-P603, M1-K602, M1-Q601, M1-R600, M1-R599,M1-R598, M1-V597, M1-S596, M1-Y595, M1-V594, M1-Q593, M1-D592, M1-G591,M1-C590, M1-T589, M1-P588, M1-L587, M1-Q586, M1-S585, M1-C584, M1-S583,M1-Y582, M1-A581, M1-S580, M1-Y579, M1-S578, M1-A577, M1-S576, M1-G575,M1-G574, M1-Y573, M1-I572, M1-A571, M1-S570, M1-A569, M1-S568, M1-Y567,M1-F566, M1-H565, M1-S564, M1-S563, M1-E562, M1-T561, M1-A560, M1-F559,M1-Y558, M1-W557, M1-S556, M1-S555, M1-T554, M1-L553, M1-S552, M1-P551,M1-T550, M1-S549, M1-T548, M1-Q547, M1-P546, M1-A545, M1-L544, M1-I543,M1-D542, M1-S541, M1-H540, M1-W539, M1-G538, M1-K537, M1-L536, M1-G535,M1-L534, M1-G533, M1-A532, M1-S531, M1-K530, M1-T529, M1-L528, M1-H527,M1-Q526, M1-Q525, M1-S524, M1-T523, M1-S522, M1-L521, M1-G520, M1-F519,M1-L518, M1-F517, M1-S516, M1-T515, M1-H514, M1-Y513, M1-N512, M1-D511,M1-E510, M1-V509, M1-S508, M1-G507, M1-S506, M1-R505, M1-H504, M1-L503,M1-P502, M1-S501, M1-L500, M1-L499, M1-S498, M1-R497, M1-Q496, M1-A495,M1-T494, M1-G493, M1-S492, M1-S491, M1-S490, M1-T489, M1-R488, M1-V487,M1-S486, M1-H485, M1-L484, M1-R483, M1-K482, M1-S481, M1-Q480, M1-S479,M1-D478, M1-S477, M1-P476, M1-R475, M1-A474, M1-T473, M1-Q472, M1-L471,M1-K470, M1-K469, M1-P468, M1-I467, M1-S466, M1-A465, M1-E464, M1-E463,M1-K462, M1-D461, M1-P460, M1-S459, M1-T458, M1-E457, M1-P456, M1-T455,M1-Q454, M1-E453, M1-S452, M1-L451, M1-E450, M1-Q449, M1-V448, M1-P447,M1-S446, M1-F445, M1-Q444, M1-C443, M1-L442, M1-K441, M1-N440, M1-T439,M1-G438, M1-D437, M1-L436, M1-T435, M1-T434, M1-S433, M1-P432, M1-K431,M1-Y430, M1-Y429, M1-E428, M1-L427, M1-A426, M1-D425, M1-E424, M1-S423,M1-S422, M1-S421, M1-F420, M1-G419, M1-H418, M1-L417, M1-S416, M1-A415,M1-A414, M1-M413, M1-S412, M1-A411, M1-S410, M1-Y409, M1-S408, M1-V407,M1-S406, M1-K405, M1-I404, M1-D403, M1-L402, M1-S401, M1-F400, M1-S399,M1-R398, M1-K397, M1-L396, M1-K395, M1-N394, M1-S393, M1-D392, M1-E391,M1-L390, M1-R389, M1-D388, M1-A387, M1-S386, M1-L385, M1-H384, M1-L383,M1-G382, M1-S381, M1-L380, M1-A379, M1-Q378, M1-V377, M1-L376, M1-P375,M1-S374, M1-D373, M1-E372, M1-L371, M1-L370, M1-S369, M1-P368, M1-Q367,M1-V366, M1-S365, M1-P364, M1-V363, M1-S362, M1-P361, M1-V360, M1-S359,M1-A358, M1-P357, M1-H356, M1-V355, M1-P354, M1-R353, M1-Q352, M1-G351,M1-A350, M1-A349, M1-E348, M1-S347, M1-T346, M1-A345, M1-S344, M1-D343,M1-A342, M1-C341, M1-P340, M1-P339, M1-S338, M1-L337, M1-P336, M1-T335,M1-E334, M1-S333, M1-K332, M1-Q331, M1-G330, M1-G329, M1-E328, M1-S327,M1-V326, M1-A325, M1-P324, M1-V323, M1-P322, M1-E321, M1-N320, M1-P319,M1-K318, M1-E317, M1-L316, M1-P315, M1-L314, M1-L313, M1-K312, M1-L311,M1-K310, M1-S309, M1-K308, M1-P307, M1-G306, M1-S305, M1-A304, M1-G303,M1-T302, M1-Q301, M1-N300, M1-K299, M1-I298, M1-K297, M1-K296, M1-E295,M1-Y294, M1-A293, M1-L292, M1-L291, M1-Q290, M1-G289, M1-L288, M1-F287,M1-N286, M1-F285, M1-N284, M1-P283, M1-S282, M1-I281, M1-T280, M1-P279,M1-R278, M1-K277, M1-E276, M1-K275, M1-V274, M1-F273, M1-R272, M1-Y271,M1-A270, M1-E269, M1-D268, M1-L267, M1-S266, M1-M265, M1-D264, M1-M263,M1-R262, M1-K261, M1-M260, M1-I259, M1-Y258, M1-A257, M1-I256, M1-A255,M1-I254, M1-T253, M1-A252, M1-S251, M1-R250, M1-S249, M1-I248, M1-G247,M1-A246, M1-L245, M1-C244, M1-H243, M1-V242, M1-L241, M1-V240, M1-C239,M1-G238, M1-N237, M1-S236, M1-A235, M1-K234, M1-A233, M1-K232, M1-E231,M1-I230, M1-F229, M1-D228, M1-V227, M1-S226, M1-K225, M1-D224, M1-L223,M1-W222, M1-P221, M1-L220, M1-I219, M1-K218, M1-E217, M1-C216, M1-F215,M1-S214, M1-D213, M1-N212, M1-V211, M1-P210, M1-V209, M1-R208, M1-L207,M1-F206, M1-H205, M1-S204, M1-E203, M1-P202, M1-I201, M1-F200, M1-D199,M1-P198, M1-K197, M1-P196, M1-C195, M1-T194, M1-N193, M1-S192, M1-A191,M1-N190, M1-L189, M1-V188, M1-Y187, M1-G186, M1-I185, M1-G184, M1-N183,M1-Q182, M1-Q181, M1-M180, M1-L179, M1-E178, M1-K177, M1-N176, M1-L175,M1-V174, M1-D173, M1-R172, M1-Q171, M1-C170, M1-G169, M1-L168, M1-Y167,M1-L166, M1-N165, M1-P164, M1-L163, M1-I162, M1-R161, M1-T160, M1-P159,M1-G158, M1-I157, M1-N156, M1-A155, M1-V154, M1-P153, M1-L152, M1-C151,M1-P150, M1-Q149, M1-S148, M1-I147, M1-C146, M1-T145, M1-P144, M1-V143,M1-L142, M1-T141, M1-S140, M1-K139, M1-G138, M1-E137, M1-C136, M1-L135,M1-G134, M1-P133, M1-F132, M1-C131, M1-R130, M1-S129, M1-F128, M1-E127,M1-A126, M1-F125, M1-G124, M1-G123, M1-A122, M1-L121, M1-L120, M1-H119,M1-V118, M1-S117, M1-N116, M1-F115, M1-S114, M1-K113, M1-E112, M1-L111,M1-K110, M1-G109, M1-L108, M1-L107, M1-V106, M1-T105, M1-L104, M1-F103,M1-C102, M1-D101, M1-S100, M1-S99, M1-L98, M1-S97, M1-A96, M1-V95,M1-D94, M1-Q93, M1-S92, M1-S91, M1-Q90, M1-D89, M1-Y88, M1-V87, M1-V86,M1-V85, M1-K84, M1-Q83, M1-S82, M1-C81, M1-D80, M1-I79, M1-D78, M1-V77,M1-K76, M1-H75, M1-K74, M1-A73, M1-S72, M1-H71, M1-Q70, M1-I69, M1-L68,M1-E67, M1-T66, M1-I65, M1-L64, M1-V63, M1-K62, M1-D61, M1-Q60, M1-Q59,M1-L58, M1-R57, M1-R56, M1-K55, M1-M54, M1-L53, M1-K52, M1-S51, M1-C50,M1-N49, M1-I48, M1-N47, M1-I46, M1-A45, M1-E44, M1-L43, M1-I42, M1-H41,M1-S40, M1-T39, M1-N38, M1-Y37, M1-E36, M1-V35, M1-F34, M1-P33, M1-R32,M1-S31, M1-D30, M1-I29, M1-L28, M1-L27, M1-V26, M1-K25, M1-E24, M1-T23,M1-G22, M1-S21, M1-E20, M1-L19, M1-L18, M1-A17, M1-V16, M1-L15, M1-R14,M1-E13, M1-T12, M1-V11, M1-I10, M1-Q9, M1-T8, and/or M1-G7 of SEQ IDNO:42. Polynucleotide sequences encoding these polypeptides are alsoprovided. The present invention also encompasses the use of the humanBMY_HPP5 phosphatase C-terminal deletion polypeptides as immunogenicand/or antigenic epitopes as described elsewhere herein.

The present invention also encompasses immunogenic and/or antigenicepitopes of the human BMY_HPP5 phosphatase polypeptide.

The human phosphatase polypeptides of the present invention weredetermined to comprise several phosphorylation sites based upon theMotif algorithm (Genetics Computer Group, Inc.). The phosphorylation ofsuch sites may regulate some biological activity of the humanphosphatase polypeptide. For example, phosphorylation at specific sitesmay be involved in regulating the proteins ability to associate or bindto other molecules (e.g., proteins, ligands, substrates, DNA, etc.). Inthe present case, phosphorylation may modulate the ability of the humanphosphatase polypeptide to associate with other polypeptides,particularly cognate ligand for human phosphatase, or its ability tomodulate certain cellular signal pathways.

Specifically, the BMY_HPP5 polypeptide was predicted to comprise onetyrosine phosphorylation site using the Motif algorithm (GeneticsComputer Group, Inc.). Such sites are phosphorylated at the tyrosineamino acid residue. The consensus pattern for tyrosine phosphorylationsites are as follows: [RK]-x(2)-[DE]-x(3)-Y, or [RK]-x(3)-[DE]-x(2)-Y,where Y represents the phosphorylation site and ‘x’ represents anintervening amino acid residue. Additional information specific totyrosine phosphorylation sites can be found in Patschinsky T., HunterT., Esch F. S., Cooper J. A., Sefton B. M., Proc. Natl. Acad. Sci.U.S.A. 79:973-977 (1982); Hunter T., J. Biol. Chem. 257:4843-4848(1982), and Cooper J. A., Esch F. S., Taylor S. S., Hunter T., J. Biol.Chem. 259:7835-7841 (1984), which are hereby incorporated herein byreference.

In preferred embodiments, the following tyrosine phosphorylation sitepolypeptides are encompassed by the present invention:NGCVLVHCLAGISRSATIAIAYI (SEQ ID NO:103). Polynucleotides encoding thesepolypeptides are also provided. The present invention also encompassesthe use of the human BMY_HPP5 tyrosine phosphorylation site polypeptidesas immunogenic and/or antigenic epitopes as described elsewhere herein.

The human phosphatase polypeptide was predicted to comprise twelve PKCphosphorylation sites using the Motif algorithm (Genetics ComputerGroup, Inc.). In vivo, protein kinase C exhibits a preference for thephosphorylation of serine or threonine residues. The PKC phosphorylationsites have the following consensus pattern: [ST]-x-[RK], where S or Trepresents the site of phosphorylation and ‘x’ an intervening amino acidresidue. Additional information regarding PKC phosphorylation sites canbe found in Woodget J. R., Gould K. L., Hunter T., Eur. J. Biochem.161:177-184 (1986), and Kishimoto A., Nishiyama K., Nakanishi H.,Uratsuji Y., Nomura H., Takeyama Y., Nishizuka Y., J. Biol. Chem.260:12492-12499 (1985); which are hereby incorporated by referenceherein.

In preferred embodiments, the following PKC phosphorylation sitepolypeptides are encompassed by the present invention: GTQIVTERLVALL(SEQ ID NO:91), LLESGTEKVLLID (SEQ ID NO:92), ELIQHSAKHKVDI (SEQ IDNO:93), VDIDCSQKVVVYD (SEQ ID NO:94), DRLEDSNKLKRSF (SEQ ID NO:95),TTLDGTNKLCQFS (SEQ ID NO:96), PKKLQTARPSDSQ (SEQ ID NO: 97),PSDSQSKRLHSVR (SEQ ID NO:98), SKRLHSVRTSSSG (SEQ ID NO:99),GDQVYSVRRRQKP (SEQ ID NO:100), RRQKPSDRADSRR (SEQ ID NO:101), and/orSDRADSRRSWHEE (SEQ ID NO:102). Polynucleotides encoding thesepolypeptides are also provided. The present invention also encompassesthe use of the human BMY_HPP5 phosphatase PKC phosphorylation sitepolypeptides as immunogenic and/or antigenic epitopes as describedelsewhere herein.

The human phosphatase polypeptide has been shown to comprise sixglycosylation sites according to the Motif algorithm (Genetics ComputerGroup, Inc.). As discussed more specifically herein, proteinglycosylation is thought to serve a variety of functions including:augmentation of protein folding, inhibition of protein aggregation,regulation of intracellular trafficking to organelles, increasingresistance to proteolysis, modulation of protein antigenicity, andmediation of intercellular adhesion.

Asparagine phosphorylation sites have the following consensus pattern,N-{P}-[ST]-{P}, wherein N represents the glycosylation site. However, itis well known that that potential N-glycosylation sites are specific tothe consensus sequence Asn-Xaa-Ser/Thr. However, the presence of theconsensus tripeptide is not sufficient to conclude that an asparagineresidue is glycosylated, due to the fact that the folding of the proteinplays an important role in the regulation of N-glycosylation. It hasbeen shown that the presence of proline between Asn and Ser/Thr willinhibit N-glycosylation; this has been confirmed by a recent statisticalanalysis of glycosylation sites, which also shows that about 50% of thesites that have a proline C-terminal to Ser/Thr are not glycosylated.Additional information relating to asparagine glycosylation may be foundin reference to the following publications, which are herebyincorporated by reference herein: Marshall R. D., Annu. Rev. Biochem.41:673-702 (1972); Pless D. D., Lennarz W. J., Proc. Natl. Acad. Sci.U.S.A. 74:134-138 (1977); Bause E., Biochem. J. 209:331-336 (1983);Gavel Y., von Heijne G., Protein Eng. 3:433-442 (1990); and Miletich J.P., Broze G. J. Jr., J. Biol. Chem. 265:11397-11404 (1990).

In preferred embodiments, the following asparagine glycosylation sitepolypeptides are encompassed by the present invention: PFVEYNTSHILEAI(SEQ ID NO:85), EAININCSKLMKRR (SEQ ID NO:86), IGYVLNASNTCPKP (SEQ IDNO:87), LRVPVNDSFCEKIL (SEQ ID NO:88), EKKIKNQTGASGPK (SEQ ID NO:89),and/or SIMSENRSREELGK (SEQ ID NO:90). Polynucleotides encoding thesepolypeptides are also provided. The present invention also encompassesthe use of the human BMY_HPP5 phosphatase asparagine glycosylation sitepolypeptides as immunogenic and/or antigenic epitopes as describedelsewhere herein.

The present invention encompasses the use of BMY_HPP5 inhibitors and/oractivators of BMY_HPP5 activity for the treatment, detection,amelioration, or prevention of phosphatase associated disorders,including but not limited to metabolic diseases such as diabetes, inaddition to neural and/or cardiovascular diseases and disorders. Thepresent invention also encompasses the use of BMY_HPP5 inhibitors and/oractivators of BMY_HPP5 activity as immunosuppressive agents,anti-inflammatory agents, and/or anti-tumor agents.

The present invention encompasses the use of BMY_HPP5 phosphataseinhibitors, including, antagonists such as antisense nucleic acids, inaddition to other antagonists, as described herein, in a therapeuticregimen to diagnose, prognose, treat, ameliorate, and/or preventdiseases where a kinase activity is insufficient. One, non-limitingexample of a disease which may occur due to insufficient kinase activityare certain types of diabetes, where one or more kinases involved in theinsulin receptor signal pathway may have insufficient activity orinsufficient expression, for example.

Moreover, the present invention encompasses the use of BMY_HPP5phosphatase activators, and/or the use of the BMY_HPP5 phosphatase geneor protein in a gene therapy regimen, as described herein, for thediagnoses, prognoses, treatment, amelioration, and/or prevention ofdiseases and/or disorders where a kinase activity is overly high, suchas a cancer where a kinase oncogene product has excessive activity orexcessive expression.

The present invention also encompasses the use of catalytically inactivevariants of BMY_HPP5 proteins, including fragments thereof, such as aprotein therapeutic, or the use of the encoding polynucleotide sequenceor as gene therapy, for example, in the diagnoses, prognosis, treatment,amelioration, and/or prevention of diseases or disorders wherephosphatase activity is overly high.

The present invention encompasses the use of antibodies directed againstthe BMY_HPP5 polypeptides, including fragment and/or variants thereof,of the present invention in diagnostics, as a biomarkers, and/or as atherapeutic agents.

The present invention encompasses the use of an inactive, non-catalytic,mutant of the BMY_HPP5 phosphatase as a substrate trapping mutant tobind cellular phosphoproteins or a library of phosphopeptides toidentify substrates of the BMY_HPP5 polypeptides.

The present invention encompasses the use of the BMY_HPP5 polypeptides,to identify inhibitors or activators of the BMY_HPP5 phosphataseactivity using either in vitro or ‘virtual’ (in silico) screeningmethods.

One embodiment of the invention relates to a method for identifying acompound as an activator or inhibitor of the BMY_HPP5 phosphatasecomprising the steps of: i.) contacting a BMY_HPP5 phosphatase inhibitoror activator labeled with an analytically detectable reagent with theBMY_HPP5 phosphatase under conditions sufficient to form a complex withthe inhibitor or activator; ii.) contacting said complex with a samplecontaining a compound to be identified; iii) and identifying thecompound as an inhibitor or activator by detecting the ability of thetest compound to alter the amount of labeled known BMY_HPP5 phosphataseinhibitor or activator in the complex.

Another embodiment of the invention relates to a method for identifyinga compound as an activator or inhibitor of a BMY_HPP5 phosphatasecomprising the steps of: i.) contacting the BMY_HPP5 phosphatase with acompound to be identified; and ii.) and measuring the ability of theBMY_HPP5 phosphatase to remove phosphate from a substrate.

The present invention also encomposses a method for identifying a ligandfor the BMY_HPP5 phosphatase comprising the steps of: i.) contacting theBMY_HPP5 phosphatase with a series of compounds under conditions topermit binding; and ii.) detecting the presence of any ligand-boundprotein.

Preferably, the above referenced methods comprise the BMY_HPP5phosphatase in a form selected from the group consisting of whole cells,cytosolic cell fractions, membrane cell fractions, purified or partiallypurified forms. The invention also relates to recombinantly expressedBMY_HPP5 phosphatase in a purified, substantially purified, orunpurified state. The invention further relates to BMY_HPP5 phosphatasefused or conjugated to a protein, peptide, or other molecule or compoundknown in the art, or referenced herein.

The present invention also encompasses a pharmaceutical composition ofthe BMY_HPP5 phosphatase polypeptide comprising a compound identified byabove referenced methods and a pharmaceutically acceptable carrier.

In preferred embodiments, the present invention encompasses apolynucleotide lacking the initiating start codon, in addition to, theresulting encoded polypeptide of BMY_HPP5. Specifically, the presentinvention encompasses the polynucleotide corresponding to nucleotides473 thru 2464 of SEQ ID NO:41, and the polypeptide corresponding toamino acids 2 thru 665 of SEQ ID NO:42. Also encompassed are recombinantvectors comprising said encoding sequence, and host cells comprisingsaid vector.

The present invention also provides a three-dimensional homology modelof the BMY_HPP5 polypeptide (see FIG. 38) representing amino acids N157to 1300 of BMY_HPP5 (SEQ ID NO:42). A three-dimensional homology modelcan be constructed on the basis of the known structure of a homologousprotein (Greer et al, 1991, Lesk, et al, 1992, Cardozo, et al, 1995,Yuan, et al, 1995). The homology model of the BMY_HPP5 polypeptide,corresponding to amino acid residues N157 to 1300 of SEQ ID NO:42, wasbased upon the homologous structure of 1vhr from the N-terminus of humandual specificity phosphatase MAP Kinase phosphatase 3 (also calledPYST1) (residues A204-L347; Protein Data Bank, PDB entry 1 mkp chain AGenbank Accession No. gi|5822131; SEQ ID NO:208) (Stewart, A. E., etal., 1999) and is defined by the set of structural coordinates set forthin Table X herein.

Homology models are useful when there is no experimental informationavailable on the protein of interest. A 3-dimensional model can beconstructed on the basis of the known structure of a homologous protein(Greer et al, 1991, Lesk, et al, 1992, Cardozo, et al, 1995, Sali, etal, 1995).

Those of skill in the art will understand that a homology model isconstructed on the basis of first identifying a template, or, protein ofknown structure which is similar to the protein without known structure.This can be accomplished by through pairwise alignment of sequencesusing such programs as FASTA (Pearson, et al 1990) and BLAST (Altschul,et al, 1990). In cases where sequence similarity is high (greater than30%) these pairwise comparison methods may be adequate. Likewise,multiple sequence alignments or profile-based methods can be used toalign a query sequence to an alignment of multiple (structurally andbiochemically) related proteins. When the sequence similarity is low,more advanced techniques are used such as fold recognition (proteinthreading; Hendlich, et al, 1990), where the compatibility of aparticular sequence with the 3-dimensional fold of a potential templateprotein is gauged on the basis of a knowledge-based potential. Followingthe initial sequence alignment, the query template can be optimallyaligned by manual manipulation or by incorporation of other features(motifs, secondary structure predictions, and allowed sequenceconservation). Next, structurally conserved regions can be identifiedand used to construct the core secondary structure (Sali, et al, 1995).Loops can be added using knowledge-based techniques, and refinedperforming force field calculations (Sali, et al, 1995; Cardozo, et al,1995).

For BMY_HPP5 the pairwise alignment method FASTA (Pearson, et al 1990)and fold recognition methods (protein threading) generated identicalsequence alignments for a portion (residues N157 to 1300 of SEQ IDNO:42) of BMY_HPP5 aligned with the sequence of the human dualspecificity phosphatase MAP Kinase phosphatase 3 (also called PYST1)(residues A204-L347; Protein Data Bank, PDB entry 1 mkp chain A; GenbankAccession No. gi|5822131; SEQ ID NO:208) (Stewart, A. E., et al., 1999).The alignment of BMY_HPP5 with PDB entry 1 mkp is set forth in FIG. 37.In this invention, the homology model of BMY_HPP5 was derived from thesequence alignment set forth in FIG. 37, and thence an overall atomicmodel including plausible sidechain orientations using the program LOOK(Levitt, 1992). The three dimensional model for BMY_HPP5 is defined bythe set of structure coordinates as set forth in Table X and visualizedin FIG. 38.

In order to recognize errors in three-dimensional structures knowledgebased mean fields can be used to judge the quality of protein folds(Sippl 1993). The methods can be used to recognize misfolded structuresas well as faulty parts of structural models. The technique generates anenergy graph where the energy distribution for a given protein fold isdisplayed on the y-axis and residue position in the protein fold isdisplayed on the x-axis. The knowledge based mean fields compose a forcefield derived from a set of globular protein structures taken as asubset from the Protein Data Bank (Bernstein et. al. 1977). To analyzethe quality of a model the energy distribution is plotted and comparedto the energy distribution of the template from which the model wasgenerated. FIG. 39 shows the energy graph for the BMY_HPP5 model (dottedline) and the template (1 mkp, a dual-specificity phosphatase) fromwhich the model was generated. It is clear that the model and templatehave similar energies over the aligned region, suggesting that BMY_HPP5is in a “native-like” conformation. This graph supports the motif andsequence alignments in confirming that the three dimensional structurecoordinates of BMY_HPP5 are an accurate and useful representation forthe polypeptide.

The term “structure coordinates” refers to Cartesian coordinatesgenerated from the building of a homology model.

Those of skill in the art will understand that a set of structurecoordinates for a protein is a relative set of points that define ashape in three dimensions. Thus, it is possible that an entirelydifferent set of coordinates could define a similar or identical shape.Moreover, slight variations in the individual coordinates, as emanatefrom generation of similar homology models using different alignmenttemplates (i.e., other than the structure coordinates of 1 mkp), and/orusing different methods in generating the homology model, will haveminor effects on the overall shape. Variations in coordinates may alsobe generated because of mathematical manipulations of the structurecoordinates. For example, the structure coordinates set forth in Table Xand visualized in FIG. 38 could be manipulated by fractionalization ofthe structure coordinates; integer additions or subtractions to sets ofthe structure coordinates, inversion of the structure coordinates or anycombination of the above.

Various computational analyses are therefore necessary to determinewhether a molecule or a portion thereof is sufficiently similar to allor parts of BMY_HPP5 described above as to be considered the same. Suchanalyses may be carried out in current software applications, such asINSIGHTII (Molecular Simulations Inc., San Diego, Calif.) version 2000and as described in the accompanying User's Guide.

Using the superimposition tool in the program INSIGHTII comparisons canbe made between different structures and different conformations of thesame structure. The procedure used in INSIGHTII to compare structures isdivided into four steps: 1) load the structures to be compared; 2)define the atom equivalencies in these structures; 3) perform a fittingoperation; and 4) analyze the results. Each structure is identified by aname. One structure is identified as the target (i.e., the fixedstructure); the second structure (i.e., moving structure) is identifiedas the source structure. Since atom equivalency within INSIGHTII isdefined by user input, for the purpose of this invention we will defineequivalent atoms as protein backbone atoms (N, Cα, C and O) for allconserved residues between the two structures being compared. We willalso consider only rigid fitting operations. When a rigid fitting methodis used, the working structure is translated and rotated to obtain anoptimum fit with the target structure. The fitting operation uses analgorithm that computes the optimum translation and rotation to beapplied to the moving structure, such that the root mean squaredifference of the fit over the specified pairs of equivalent atom is anabsolute minimum. This number, given in angstroms, is reported byINSIGHTII. For the purpose of this invention, any homology model of aBMY_HPP5 that has a root mean square deviation of conserved residuebackbone atoms (N, Cα, C, O) of less than 3.0 A when superimposed on therelevant backbone atoms described by structure coordinates listed inTable X and visualized in FIG. 38 are considered identical. Morepreferably, the root mean square deviation is less than 2.0 Å.

This invention as embodied by the homology model enables thestructure-based design of modulators of the biological function ofBMY_HPP5, as well as mutants with altered biological function and/orspecificity.

There is 40% sequence identity between catalytic domain of BMY_HPP5 and1 mkp which was used as the template for 3D model generation. For theBMY_HPP5 the functionally important residues are located in a cleftcomprised of residues D213, H243, C244, R250, and S251. All theseresidues are conserved in 1 mkp (for structure determination studies thecysteine was mutated to a serine in 1 mkp). Based on the sequencealignment disclosed in FIG. 37 and the structural model disclosed inTable X and visualized in FIG. 38, D213 is identified as a general acid,C244 as the catalytic Cysteine nucleophile which cleaves thephosphodiester bond, and R250 as the essential Argenine which activatesthe bond for cleavage as described in the literature (reviewed by Faumanand Saper, 1996). Other important residues include F287 which impartssubstrate specificity onto the enzyme. All of these residues areconserved.

In a preferred embodiment of the present invention, the moleculecomprises the cleft region defined by structure coordinates of BMY_HPP5amino acids described above according to Table X, or a mutant of saidmolecule.

More preferred are molecules comprising all or any part of the cleft ora mutant or homologue of said molecule or molecular complex. By mutantor homologue of the molecule it is meant a molecule that has a root meansquare deviation from the backbone atoms of said BMY_HPP5 amino acids ofnot more than 3.5 Angstroms.

The term “root mean square deviation” means the square root of thearithmetic mean of the squares of the deviations from the mean. It is away to express the deviation or variation from a trend or object. Forpurposes of this invention, the “root mean square deviation” defines thevariation in the backbone of a protein from the relevant portion of thebackbone of BMY_HPP5 as defined by the structure coordinates describedherein.

The structure coordinates of a BMY_HPP5 homology model portions thereofare stored in a machine-readable storage medium. Such data may be usedfor a variety of purposes, such as drug discovery.

Accordingly, in one embodiment of this invention is provided amachine-readable data storage medium comprising a data storage materialencoded with the structure coordinates set forth in Table X.

One embodiment utilizes System 10 as disclosed in WO 98/11134, thedisclosure of which is incorporated herein by reference in its entirety.Briefly, one version of these embodiments comprises a computercomprising a central processing unit (“CPU”), a working memory which maybe, e.g, RAM (random-access memory) or “core” memory, mass storagememory (such as one or more disk drives or CD-ROM drives), one or morecathode-ray tube (“CRT”) display terminals, one or more keyboards, oneor more input lines, and one or more output lines, all of which areinterconnected by a conventional bidirectional system bus.

Input hardware, coupled to the computer by input lines, may beimplemented in a variety of ways. Machine-readable data of thisinvention may be inputted via the use of a modem or modems connected bya telephone line or dedicated data line. Alternatively or additionally,the input hardware may comprise CD-ROM drives or disk drives. Inconjunction with a display terminal, keyboard may also be used as aninput device.

Output hardware, coupled to the computer by output lines, may similarlybe implemented by conventional devices. By way of example, outputhardware may include a CRT display terminal for displaying a graphicalrepresentation of a region or domain of the present invention using aprogram such as QUANTA as described herein. Output hardware might alsoinclude a printer, so that hard copy output may be produced, or a diskdrive, to store system output for later use.

In operation, the CPU coordinates the use of the various input andoutput devices, coordinates data accesses from mass storage, andaccesses to and from the working memory, and determines the sequence ofdata processing steps. A number of programs may be used to process themachine-readable data of this invention. Such programs are discussed inreference to the computational methods of drug discovery as describedherein. Specific references to components of the hardware system areincluded as appropriate throughout the following description of the datastorage medium.

For the purpose of the present invention, any magnetic data storagemedium which can be encoded with machine-readable data would besufficient for carrying out the storage requirements of the system. Themedium could be a conventional floppy diskette or hard disk, having asuitable substrate, which may be conventional, and a suitable coating,which may be conventional, on one or both sides, containing magneticdomains whose polarity or orientation could be altered magnetically, forexample. The medium may also have an opening for receiving the spindleof a disk drive or other data storage device.

The magnetic domains of the coating of a medium may be polarized ororiented so as to encode in a manner which may be conventional, machinereadable data such as that described herein, for execution by a systemsuch as the system described herein.

Another example of a suitable storage medium which could also be encodedwith such machine-readable data, or set of instructions, which could becarried out by a system such as the system described herein, could be anoptically-readable data storage medium. The medium could be aconventional compact disk read only memory (CD-ROM) or a rewritablemedium such as a magneto-optical disk which is optically readable andmagneto-optically writable. The medium preferably has a suitablesubstrate, which may be conventional, and a suitable coating, which maybe conventional, usually of one side of substrate.

In the case of a CD-ROM, as is well known, the coating is reflective andis impressed with a plurality of pits to encode the machine-readabledata. The arrangement of pits is read by reflecting laser light off thesurface of the coating. A protective coating, which preferably issubstantially transparent, is provided on top of the reflective coating.

In the case of a magneto-optical disk, as is well known, the coating hasno pits, but has a plurality of magnetic domains whose polarity ororientation can be changed magnetically when heated above a certaintemperature, as by a laser. The orientation of the domains can be readby measuring the polarization of laser light reflected from the coating.The arrangement of the domains encodes the data as described above.

Thus, in accordance with the present invention, data capable ofdisplaying the three dimensional structure of the BMY_HPP5 homologymodel, or portions thereof and their structurally similar homologues isstored in a machine-readable storage medium, which is capable ofdisplaying a graphical three-dimensional representation of thestructure. Such data may be used for a variety of purposes, such as drugdiscovery.

For the first time, the present invention permits the use, throughhomology modeling based upon the sequence of BMY_HPP5 (FIGS. 5A-D; SEQID NO:42) of structure-based or rational drug design techniques todesign, select, and synthesize chemical entities that are capable ofmodulating the biological function of BMY_HPP5.

Accordingly, the present invention is also directed to the entiresequence in FIGS. 5A-D or any portion thereof for the purpose ofgenerating a homology model for the purpose of 3D structure-based drugdesign.

For purposes of this invention, we include mutants or homologues of thesequence in FIGS. 5A-D or any portion thereof. In a preferredembodiment, the mutants or homologues have at least 25% identity, morepreferably 50% identity, more preferably 75% identity, and mostpreferably 90% identity to the amino acid residues in FIGS. 5A-D.

The three-dimensional model structure of the BMY_HPP5 will also providemethods for identifying modulators of biological function. Variousmethods or combination thereof can be used to identify these compounds.

Structure coordinates of the catalytic region defined above can also beused to identify structural and chemical features. Identified structuralor chemical features can then be employed to design or select compoundsas potential BMY_HPP5 modulators. By structural and chemical features itis meant to include, but is not limited to, van der Waals interactions,hydrogen bonding interactions, charge interaction, hydrophobic bondinginteraction, and dipole interaction. Alternatively, or in conjunction,the three-dimensional structural model can be employed to design orselect compounds as potential BMY_HPP5 modulators. Compounds identifiedas potential BMY_HPP5 modulators can then be synthesized and screened inan assay characterized by binding of a test compound to the BMY_HPP5, orin characterizing BMY_HPP5 deactivation in the presence of a smallmolecule. Examples of assays useful in screening of potential BMY_HPP5modulators include, but are not limited to, screening in silico, invitro assays and high throughput assays. Finally, these methods may alsoinvolve modifying or replacing one or more amino acids from BMY_HPP5according to Table X.

However, as will be understood by those of skill in the art upon thisdisclosure, other structure based design methods can be used. Variouscomputational structure based design methods have been disclosed in theart.

For example, a number of computer modeling systems are available inwhich the sequence of the BMY_HPP5 and the BMY_HPP5 structure (i.e.,atomic coordinates of BMY_HPP5 and/or the atomic coordinates of theactive site as provided in Table X) can be input. This computer systemthen generates the structural details of one or more these regions inwhich a potential BMY_HPP5 modulator binds so that complementarystructural details of the potential modulators can be determined. Designin these modeling systems is generally based upon the compound beingcapable of physically and structurally associating with BMY_HPP5. Inaddition, the compound must be able to assume a conformation that allowsit to associate with BMY_HPP5. Some modeling systems estimate thepotential inhibitory or binding effect of a potential BMY_HPP5 modulatorprior to actual synthesis and testing.

Methods for screening chemical entities or fragments for their abilityto associate with a given protein target are also well known. Oftenthese methods begin by visual inspection of the binding site on thecomputer screen. Selected fragments or chemical entities are thenpositioned in one or more of the active site region in BMY_HPP5. Dockingis accomplished using software such as INSIGHTII, QUANTA and SYBYL,following by energy minimization and molecular dynamics with standardmolecular mechanic forcefields such as CHARMM and AMBER. Examples ofcomputer programs which assist in the selection of chemical fragment orchemical entities useful in the present invention include, but are notlimited to, GRID (Goodford, 1985), AUTODOCK (Goodsell, 1990), and DOCK(Kuntz et al. 1982).

Upon selection of preferred chemical entities or fragments, theirrelationship to each other and BMY_HPP5 can be visualized and thenassembled into a single potential modulator. Programs useful inassembling the individual chemical entities include, but are not limitedto SYBYL and LeapFrog (Tripos Associates, St. Louis Mo.), LUDI (Bohm1992) and 3D Database systems (Martin 1992).

Alternatively, compounds may be designed de novo using either an emptyactive site or optionally including some portion of a known inhibitor.Methods of this type of design include, but are not limited to LUDI(Bohm 1992) and LeapFrog (Tripos Associates, St. Louis Mo.).

In addition, BMY_HPP5 is overall well suited to modern methods includingcombinatorial chemistry.

Programs such as DOCK (Kuntz et al. 1982) can be used with the atomiccoordinates from the homology model to identify potential ligands fromdatabases or virtual databases which potentially bind the in the metalbinding region, and which may therefore be suitable candidates forsynthesis and testing.

Additionally, the three-dimensional homology model of BMY_HPP5 will aidin the design of mutants with altered biological activity.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO: 41 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides consisting of a nucleotide sequence described bythe general formula of a−b, where a is any integer between 1 to 5097 ofSEQ ID NO:41, b is an integer between 15 to 5111, where both a and bcorrespond to the positions of nucleotide residues shown in SEQ IDNO:41, and where b is greater than or equal to a+14.

Features of the Polypeptide Encoded by Gene No:6

The development of inflammatory disease is characterized by infiltrationof circulating blood cells across the endothelium into the tissue. Anumber of key events occur in the endothelial cells that mediate this“gateway” function. The endothelial cells express receptors andchemokines that sequentially tether the leukocytes, activate them, causethem to tightly adhere, and aid in their transmigration throughendothelial cell junctions. This process is initiated by the productionof early inflammatory mediators such as TNF-α. The coordinatedexpression of receptors and chemokines is mediated by intracellularsignaling molecules including kinases, scaffolding proteins, andtranscription factors. These molecules thus form a signaling cascadethat may be a “master switch” for the development of inflammatoryprocesses. Components of this cascade such as the transcription factorNF-kB are known. However, there are many other components that have notyet been identified. The analysis of genes that are differentiallyexpressed in TNF-α-activated endothelium may help to identify othercomponents of this “master switch” cascade.

To this end, the RNA expressed in TNF-α-stimulated human lungmicrovascular endothelial cells has been analyzed to identify geneproducts involved in regulatory events. Resting cells were stimulatedfor 1 h with TNF-α, and the RNA was isolated from the cells.Complementary DNA (cDNA) was created from the isolated RNA. The cDNAsthat were upregulated in response to TNFα were identified usingsubtractive hybridization methodology.

A novel dual specificity phosphatase (DSP), RET31 (Regulated inEndothelial cells treated with TNF-α clone 31) (FIGS. 13A-F) wasidentified from the TNF-α treated endothelial subtraction library. Thedual specificity phosphatase catalytic (DSPc) domain for RET 31 wasidentified using the DSPc PFAM-HMM (PF00782). A search for homologuesidentified three other DSPs that contain extensive homology to RET31(FIGS. 14A-C). RET31, DUS8, DUSP6 and MAP-kinase phosphatase 5 are shownin a multiple sequence alignment comparing the DSPc domains of thesefour proteins (FIG. 17).

RET31 was confirmed to be up-regulated by TNF-α, reaching a peak ofexpression at 6 h by northern blot analysis (FIG. 15). RET31 mRNA wasvirtually undetectable in brain, spleen, and peripheral blood leukocytesby Northern blot analysis.

RET31 is believed to represent a novel splice variant of the BMY_HPP5polypeptide of the present invention. The sequence for RET31 differs inthe 5′ end from that of BMY_HPP5. However, comparison of the tissueexpression of RET31 and BMY_HPP5 showed significant differentialexpression despite their significant identity. Specifically, the tissueexpression of BMY_HPP5 by PCR analysis (as described elsewhere herein)suggested that there were significant levels of RET31 in the brain. Thereason for such disparate expression profiles is unclear but may berelated to the use of separate pools of RNA or to the use of alternateprobes.

In all tissues that expressed significant levels of RET31, there was aprimary hybridizing band and a secondary band of lower molecular weight.It is not clear whether this represents splice variants of the same geneor whether there is a homologue present.

The polypeptide corresponding to this gene provided as SEQ ID NO:108(FIG. 13A-F), encoded by the polynucleotide sequence according to SEQ IDNO:109 (FIG. 13A-F), and/or encoded by the polynucleotide containedwithin the deposited clone, RET31, has significant homology at thenucleotide and amino acid level to a number of phosphatases, whichinclude, for example, the human protein-tyrosine phosphatase DUS8protein, also referred to as hVH-5 (DUS8; Genbank AccessionNo:gi|U27193; SEQ ID NO:110); the human dual specificity MAP kinaseDUSP6 protein (DUSP6; Genbank Accession No:gi|AB013382; SEQ ID NO:111);and the human map kinase phosphatase MKP-5 protein (MKP-5; GenbankAccession No:gi|AB026436; SEQ ID NO:112) as determined by BLASTP. Analignment of the human phosphatase polypeptide with these proteins isprovided in FIGS. 14A-C.

The human protein-tyrosine phosphatase DUS8 protein (also referred to ashVH-5) is thought to be a member of a subset of protein tyrosinephosphatases that regulate mitogen-activated protein kinase. Thecatalytic region of hVH-5 was expressed as a fusion protein and wasshown to hydrolyze p-nitrophenylphosphate and inactivatemitogen-activated protein kinase, thus proving that hVH-5 possessedphosphatase activity. Moreover, expression of hVH-5 transcripts wereinduced in PC12 cells upon nerve growth factor and insulin treatment ina manner characteristic of an immediate-early gene, suggesting apossible role in the signal transduction cascade (The J. Neurochem. 65(4), 1823-1833 (1995)).

The dual specificity MAP kinase DUSP6 protein is believed to beimplicated in pancreatic carcinogensis based upon its encodingpolynucleotide mapping to chromosome locus12q21, one of the regions offrequent allelic loss in pancreatic cancer, in addition to, its reducedexpressions amonst several pancreatic cancer cell lines (Cytogenet. CellGenet. 82 (3-4), 156-159 (1998)).

The human map kinase phosphatase MKP-5 protein was determined to belongto a group of dual specificity protein phosphatases that negativelyregulate members of the mitogen-activated protein kinase (MAPK)superfamily, which consists of three major subfamilies,MAPK/extracellular signal-regulated kinase (ERK), stress-activatedprotein kinase (SAPK)/c-Jun N-terminal kinase (JNK), and p38. Members ofthis group show distinct substrate specificities for MAPKs, differenttissue distribution and subcellular localization, and different modes ofinducibility of their expression by extracellular stimuli. MKP-5 wasshown to bind to p38 and SAPK/JNK, but not to MAPK/ERK, and inactivatep38 and SAPK/JNK, but not MAPK/ERK. p38 was determined to be thepreferred substrate for MKP-5. MKP-5 mRNA was widely expressed invarious tissues and organs, and its expression in cultured cells wasinducible by stress stimuli. Thus, MKP-5 is thought to represent a typeof dual specificity phosphatase specific for p38 and SAPK/JNK (J Biol.Chem., 274(28):19949-56, (1999)).

The determined nucleotide sequence of the RET31 cDNA in FIGS. 13A-F (SEQID NO:41) contains an open reading frame encoding a protein of about 665amino acid residues, with a deduced molecular weight of about 73.1 kDa.The amino acid sequence of the predicted RET31 polypeptide is shown inFIGS. 13A-F (SEQ ID NO:42). The RET31 protein shown in FIGS. 13A-F wasdetermined to share significant identity and similarity to several knownphosphatases, particularly, dual-specificity protein phosphatases.Specifically, the RET31 protein shown in FIGS. 13A-F was determined tobe about 50.3% identical and 56.8% similar to human protein-tyrosinephosphatase DUS8 protein (DUS8; Genbank Accession No:gi|U27193; SEQ IDNO:110); to be about 36.5% identical and 48.3% similar to the human dualspecificity MAP kinase DUSP6 protein (DUSP6; Genbank AccessionNo:gi|AB013382; SEQ ID NO:111); and to be about 34.3% identical and47.2% similar to the human map kinase phosphatase MKP-5 protein (MKP-5;Genbank Accession No:gi|AB026436; SEQ ID NO:112), as shown in FIG. 12.

Based upon the strong homology to members of the phosphatase proteins,the polypeptide encoded by the human RET31 phosphatase of the presentinvention is expected to share at least some biological activity withphosphatase proteins, preferably with members of the novelphosphotyrosine/dual-specificity (P-Tyr, P-Ser and P-Thr) phosphatases,particularly the novel phosphotyrosine/dual-specificity (P-Tyr, P-Serand P-Thr) phosphatases referenced herein.

The strong homology to phosphatases, particularly dual-specificityphosphatases, combined with the predominant localized expression inadrenal gland tissue suggests the human RET31 phosphatasepolynucleotides and polypeptides, including antagonists, and/orfragments thereof, may be useful for treating, diagnosing, prognosing,ameliorating, and/or preventing endocrine disorders, which include, butare mot limited to adrenocortical hyperfunction, adrenocorticalhypofunction, lethargy. Congenital adrenal hyperplasia, aberrant ACTHregulation, aberrant adrenaline regulation, disorders associated withdefects in P450C21, P450C18, P450C17, and P450C11 hydroxylases and in3-hydroxysteroid dehydrogenase (3-HSD), hirsutism, oligomenorrhea, acne,virilization, female pseudohermaphroditism, disorders associated withthe incidence of aberrant sexual characterisitics, disorders associatedwith aberrant cortisol secretion, hypertension, hypokalemia,hypogonadism, disorders associated with aberrant androgen secretion,adrenal virilism, Adrenal adenomas, Adrenal carcinomas, disordersassociated with aberrant aldosterone secretion, aldosteronism, disordersassociated with aberrant steriod biosynthesis, disorders associated withaberrant steriod transport, disorders associated with aberrant steriodsecretion, disorders associated with aberrant steriod excretion,Addison's syndrome, and Cushing's syndrome.

The strong homology to phosphatases, particularly dual-specificityphosphatases, combined with the localized expression in testis andprostate tissue suggests the human RET31 phosphatase polynucleotides andpolypeptides, including antagonists, and/or fragments thereof, may beuseful for treating, diagnosing, prognosing, and/or preventing malereproductive disorders, such as, for example, male infertility,impotence, prostate cancer, ejaculatory disorders, and/or testicularcancer. This gene product may also be useful in assays designed toidentify binding agents, as such agents (antagonists) are useful as malecontraceptive agents. The testes are also a site of active geneexpression of transcripts that is expressed, particularly at low levels,in other tissues of the body. Therefore, this gene product may beexpressed in other specific tissues or organs where it may play relatedfunctional roles in other processes, such as hematopoiesis,inflammation, bone formation, and kidney function, to name a fewpossible target indications. If fact, increased expression of certainphosphatases have been identified as tumor markers for testicular cancer(see, for example, Koshida, K., Nishino, A., Yamamoto, H., Uchibayashi,T., Naito, K., Hisazumi, H., Hirano, K., Hayashi, Y., Wahren, B.,Andersson, L, J. Urol., 146(1):57-60, (1991); and Klein, E A, Urol.Clin. North. Am., 20(1):67-73, (1993)).

The strong homology to phosphatases, particularly dual-specificityphosphatases, combined with the significant localized expression inovary and placental tissue suggests the human phosphatasepolynucleotides and polypeptides may be useful in treating, diagnosing,prognosing, and/or preventing reproductive disorders.

In preferred embodiments, RET31 polynucleotides and polypeptidesincluding agonists and fragments thereof, have uses which includetreating, diagnosing, prognosing, and/or preventing the following,non-limiting, diseases or disorders of the uterus: dysfunctional uterinebleeding, amenorrhea, primary dysmenorrhea, sexual dysfunction,infertility, pelvic inflammatory disease, endometriosis, placentalaromatase deficiency, premature menopause, and placental dysfunction.

The strong homology to phosphatases, particularly dual-specificityphosphatases, combined with the significant localized expression inskeletal tissue suggests the human phosphatase polynucleotides andpolypeptides may be useful in treating, diagnosing, prognosing, and/orpreventing muscle diseases and/or disorders, which include but are notlimited to, musculodegenerative disorders, multiple sclerosis, atrophy,ticks.

Alternatively, the strong homology to phosphatases, particularlydual-specificity phosphatases, combined with the significant localizedexpression in liver tissue suggests the human phosphatasepolynucleotides and polypeptides may be useful in treating, diagnosing,prognosing, and/or preventing hepatic diseases and/or disorders.Representative uses are described in the “Hyperproliferative Disorders”,“Infectious Disease”, and “Binding Activity” sections below, andelsewhere herein. Briefly, the protein can be used for the detection,treatment, and/or prevention of hepatoblastoma, jaundice, hepatitis,liver metabolic diseases and conditions that are attributable to thedifferentiation of hepatocyte progenitor cells, cirrhosis, hepaticcysts, pyrogenic abscess, amebic abcess, hydatid cyst,cystadenocarcinoma, adenoma, focal nodular hyperplasia, hemangioma,hepatocellulae carcinoma, cholangiocarcinoma, angiosarcoma, andgranulomatous liver disease. In addition the protein product is usefulfor treating developmental abnormalities, fetal deficiencies, pre-nataldisorders and various would-healing diseases and/or tissue trauma.

Moreover, polynucleotides and polypeptides, including fragments and/orantagonists thereof, have uses which include, directly or indirectly,treating, preventing, diagnosing, and/or prognosing the following,non-limiting, hepatic infections: liver disease caused by sepsisinfection, liver disease caused by bacteremia, liver disease caused byPneomococcal pneumonia infection, liver disease caused by Toxic shocksyndrome, liver disease caused by Listeriosis, liver disease caused byLegionnaries' disease, liver disease caused by Brucellosis infection,liver disease caused by Neisseria gonorrhoeae infection, liver diseasecaused by Yersinia infection, liver disease caused by Salmonellosis,liver disease caused by Nocardiosis, liver disease caused by Spirocheteinfection, liver disease caused by Treponema pallidum infection, liverdisease caused by Brrelia burgdorferi infection, liver disease caused byLeptospirosis, liver disease caused by Coxiella burnetii infection,liver disease caused by Rickettsia richettsii infection, liver diseasecaused by Chlamydia trachomatis infection, liver disease caused byChlamydia psittaci infection, in addition to any other hepatic diseaseand/or disorder implicated by the causative agents listed above orelsewhere herein.

The strong homology to phosphatases, particularly dual-specificityphosphatases, combined with the significant localized expression inplacental tissue suggests the human phosphatase polynucleotides andpolypeptides may be useful in treating, diagnosing, prognosing, and/orpreventing a variety of vascular disorders and conditions, whichinclude, but are not limited to miscrovascular disease, vascular leaksyndrome, aneurysm, stroke, embolism, thrombosis, coronary arterydisease, arteriosclerosis, and/or atherosclerosis. Furthermore, theprotein may also be used to determine biological activity, raiseantibodies, as tissue markers, to isolate cognate ligands or receptors,to identify agents that modulate their interactions, in addition to itsuse as a nutritional supplement. Protein, as well as, antibodiesdirected against the protein may show utility as a tumor marker and/orimmunotherapy targets for the above listed tissues.

The strong homology to phosphatases, particularly dual-specificityphosphatases, combined with the predominate localized expression inpancreas tissue suggests the human RET31 phosphatase polynucleotides andpolypeptides, including antagonists, and/or fragments thereof, may beuseful for treating, diagnosing, prognosing, and/or preventingpancreatic, in addition to metabolic and gastrointestinal disorders.

In preferred embodiments, RET31 polynucleotides and polypeptidesincluding agonists, antagonists, and fragments thereof, have uses whichinclude treating, diagnosing, prognosing, and/or preventing thefollowing, non-limiting, diseases or disorders of the pancreas: diabetesmellitus, diabetes, type 1 diabetes, type 2 diabetes, adult onsetdiabetes, indications related to islet cell transplantation, indicationsrelated to pancreatic transplantation, pancreatitis, pancreatic cancer,pancreatic exocrine insufficiency, alcohol induced pancreatitis,maldigestion of fat, maldigestion of protein, hypertriglyceridemia,vitamin b12 malabsorption, hypercalcemia, hypocalcemia, hyperglycemia,ascites, pleural effusions, abdominal pain, pancreatic necrosis,pancreatic abscess, pancreatic pseudocyst, gastrinomas, pancreatic isletcell hyperplasia, multiple endocrine neoplasia type 1 (men 1) syndrome,insulitis, amputations, diabetic neuropathy, pancreatic auto-immunedisease, genetic defects of -cell function, HNF-1 aberrations (formerlyMODY3), glucokinase aberrations (formerly MODY2), HNF-4 aberrations(formerly MODY1), mitochondrial DNA aberrations, genetic defects ininsulin action, type a insulin resistance, leprechaunism,Rabson-Mendenhall syndrome, lipoatrophic diabetes, pancreatectomy,cystic fibrosis, hemochromatosis, fibrocalculous pancreatopathy,endocrinopathies, acromegaly, Cushing's syndrome, glucagonoma,pheochromocytoma, hyperthyroidism, somatostatinoma, aldosteronoma, drug-or chemical-induced diabetes such as from the following drugs: Vacor,Pentamdine, Nicotinic acid, Glucocorticoids, Thyroid hormone, Diazoxide,Adrenergic agonists, Thiazides, Dilantin, and Interferon, pancreaticinfections, congential rubella, cytomegalovirus, uncommon forms ofimmune-mediated diabetes, “stiff-man” syndrome, anti-insulin receptorantibodies, in addition to other genetic syndromes sometimes associatedwith diabetes which include, for example, Down's syndrome, Klinefelter'ssyndrome, Turner's syndrome, Wolfram's syndrome, Friedrich's ataxia,Huntington's chorea, Lawrence Moon Beidel syndrome, Myotonic dystrophy,Porphyria, and Prader Willi syndrome, and/or Gestational diabetesmellitus (GDM).

The strong homology to phosphatases, particularly dual-specificityphosphatases, combined with the predominate localized expression inthymus tissue suggests the human RET31 phosphatase polynucleotides andpolypeptides, including antagonists, and/or fragments thereof, may beuseful for treating, diagnosing, prognosing, and/or preventing immuneand hematopoietic disorders. Representative uses are described in the“Immune Activity”, “Chemotaxis”, and “Infectious Disease” sectionsbelow, and elsewhere herein. Briefly, the strong expression in immunetissue indicates a role in regulating the proliferation; survival;differentiation; and/or activation of hematopoietic cell lineages,including blood stem cells.

The RET31 polypeptide may also be useful as a preventative agent forimmunological disorders including arthritis, asthma, immunodeficiencydiseases such as AIDS, leukemia, rheumatoid arthritis, granulomatousdisease, inflammatory bowel disease, sepsis, acne, neutropenia,neutrophilia, psoriasis, hypersensitivities, such as T-cell mediatedcytotoxicity; immune reactions to transplanted organs and tissues, suchas host-versus-graft and graft-versus-host diseases, or autoimmunitydisorders, such as autoimmune infertility, lense tissue injury,demyelination, systemic lupus erythematosis, drug induced hemolyticanemia, rheumatoid arthritis, Sjogren's disease, and scleroderma.Moreover, the protein may represent a secreted factor that influencesthe differentiation or behavior of other blood cells, or that recruitshematopoietic cells to sites of injury. Thus, this gene product may beuseful in the expansion of stem cells and committed progenitors ofvarious blood lineages, and in the differentiation and/or proliferationof various cell types.

The RET31 polypeptide may be useful for modulating cytokine production,antigen presentation, or other processes, such as for boosting immuneresponses, etc. Expression in cells of lymphoid origin, indicates thenatural gene product would be involved in immune functions.

Moreover, the protein may represent a secreted factor that influencesthe differentiation or behavior of other blood cells, or that recruitshematopoietic cells to sites of injury. Thus, this gene product isthought to be useful in the expansion of stem cells and committedprogenitors of various blood lineages, and in the differentiation and/orproliferation of various cell types. Furthermore, the protein may alsobe used to determine biological activity, raise antibodies, as tissuemarkers, to isolate cognate ligands or receptors, to identify agentsthat modulate their interactions, in addition to its use as anutritional supplement. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

The human phosphatase polynucleotides and polypeptides, includingfragments and agonists thereof, may have uses which include, eitherdirectly or indirectly, for boosting immune responses.

The strong homology to phosphatases, particularly dual-specificityphosphatases, suggests the human phosphatase polynucleotides andpolypeptides may be useful in treating, diagnosing, prognosing, and/orpreventing a variety of disorders and conditions, particularlyinflammatory conditions, which include, but are not limited torheumatoid arthritis, juvenile arthritis, psoriasis, asthma,ischemia-repurfusion, multiple sclerosis, rejection of organ or tissuetransplants, chronic obstructive pulmonary disease, inflammatory boweldisease, Chrohn's disease, ulcerative colitis, inacute respiratorydistress syndrome, systemic lupus erythematosis, cystic fibrosis,autoimmune diseases, cancers, tumors, and neoplasms.

The human phosphatase polynucleotides and polypeptides, includingfragments and/or antagonists thereof, may have uses which includeidentification of modulators of human phosphatase function includingantibodies (for detection or neutralization), naturally-occurringmodulators and small molecule modulators. Antibodies to domains of thehuman phosphatase protein could be used as diagnostic agents ofcardiovascular and inflammatory conditions in patients, are useful inmonitoring the activation of signal transduction pathways, and can beused as a biomarker for the involvement of phosphatases in diseasestates, and in the evaluation of inhibitors of phosphatases in vivo.

Human phosphatase polypeptides and polynucleotides have additional useswhich include diagnosing diseases related to the over and/or underexpression of human phosphatase by identifying mutations in the humanphosphatase gene by using human phosphatase sequences as probes or bydetermining human phosphatase protein or mRNA expression levels. Humanphosphatase polypeptides may be useful for screening compounds thataffect the activity of the protein. Human phosphatase peptides can alsobe used for the generation of specific antibodies and as bait in yeasttwo hybrid screens to find proteins the specifically interact with humanphosphatase (described elsewhere herein).

Immunohistochemistry analysis of the protein localization of the RET31polypeptide (see Example 58) in normal and diseased tissues determinedthat RET31 was strongly expressed in normal respiratory epithelial cellbodies, type I and II pneumocytes, lung neutrophils, lung mast cells,lung macrophages, in comparison to the same in asthmatic patients whichshowed less staining. These results suggest that RET31 polypeptides andpolynucleotides, including fragments thereof, may be useful for thetreatment of pulmonary disorders. The decreased staining in diseasedlung tissues suggests RET31 is essential for normal cell maintainanceand homeostasis, and is downregulated in transformed, or rapidlyproliferating cells. Thus, agonists of RET31 polypeptides andpolynucleotides may be particularly useful for the treatment ofpulmonary disorders.

Immunohistochemistry analysis of the protein localization of the RET31polypeptide (see Example 58) in normal and diseased tissues determinedthat RET31 was also strongly expressed in chondrocytes and rimmingosteoblasts in degenerative arthritis, in addition to hematopoeitic celltissue. Moreover, melanocytes were strongly positive, as was skin withpsoriasis. These results suggest that RET31 may be involved ininflammatory responses of certain diseases and/or disorders. Thus, RET31polypeptides and polynucleotides, including fragments thereof, may beuseful for the treatment of inflammatory disorders, particularlyinflammatory disorders of the skin and bone, such as, psoriasis andarthritis, for example. Moreover, antagonists of RET31 polypeptides andpolynucleotides may be useful for the treatment of inflammatorydisorders, particularly inflammatory disorders of the skin and bone,such as, psoriasis and arthritis, for example.

Assays designed to assess the phosphatase activity of the RET31polypeptide have been performed and prove that RET31 does indeed havephosphatase activity as described in Example 57 herein (see FIG. 36).The observed phosphatase activity was specific to RET31 as GST alone didnot result in any observed activity. In addition, the observedphosphatase activity was specifically inhibited by the known phosphataseactive site inhibitor, vanadate.

In addition to assaying the full-length RET31 polypeptide (SEQ IDNO:109), a C-terminal deletion of RET31 was also assayed correspondingto amino acids M1 to T302 of SEQ ID NO:109). The M1 to T302 deletionmutant had an unexpected five fold increase in phosphatase activityrelative to the full-length protein.

A phosphatase with a sequence similar to the RET31 polypeptide has beenreported as MKP7 (Masuda et al., JBC 276, 39002-39011; and Tanoue etal., JBC., 276, 26269-26639). These authors reported that thephosphatase could bind to and dephosphorylate the p38 kinase and the Jnkkinase in cells, resulting in the inactivation of these kinases.Activation of p38 kinase is known to be important in the induction ofapoptosis (Herlaar and Brown, Molecular Medicine Today 5, 439-447). Onepathway where p38 has been reported to be important is in paclitaxel(Taxol®) induced apoptosis in tumor cells (Seidman et al., ExperimentalCell Research 268, 84-92). Similarly, activation of the Jnk kinase hasalso been reported to be important in the induction of apoptosis (Changand Karin, Nature 410, 37-40), including in paclitaxel induced apoptosis(Lee et al., JBC., 273, 28253-28260). Therefore, inhibitors of RET31should induce apoptosis in tumor cells by increasing the activation ofp38 and Jnk kinases in the cells by preventing the dephosphorylation ofthese kinases. This would be particularly advantageous when combinedwith a chemotherapeutic drug, such as paclitaxel, that activates p38and/or Jnk kinases to help induce apoptosis. Such a use represents anovel utility of RET31 antagonists and which has not be appreciated byMasuda et al., nor by Tanoue et al. Indeed, Masuda et al. teach thatMKP7 may be a tumor suppressor gene, in which case inhibition of MKP7would increase malignancies, which teaches away from our intended usefor RET31 inhibitors.

In preferred embodiments, the present invention encompasses the use ofinhibitors of RET31 for the treatment of cancer. Per the teachingsdescribed herein, inhibitors of RET31 may include small moleculeinhibitors of RET31 activity, inhibitors that prevent RET31 from bindingto p38 and/or Jnk kinases, antisense oligonucleotides to RET31, andantibodies directed against RET31. Such RET31 inhibitors would beparticularly useful in malignancies where RET31 was overexpressedrelative to normal tissues. In addition to the use of RET31 inhibitorsas single agents, inhibitors of RET31 would be of particular use incombination with paclitaxel and other chemotherapeutic agents thatinduce Jnk and/or p38 dependent apoptosis in tumor cells for thetreatment of malignancies. Other chemotherapeutic agents that may inducethe activation of Jnk and/or p38 leading to apoptosis that would be ofuse in combination with inhibitors of RET31 include but are not limitedto RRR-alpha-tocopherol succinate, DA-125[(8S,10S)-8-(3-aminopropanoyloxyacetyl)-10-[(2,6-dideoxy-2-fluoro-alpha-L-talopyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-1-methoxy-5,12-naphthacene-dionehydrochloride] a novel anthracycline derivative, cisplatin, tamoxifen,sulindac sulfone, sulindac, arsenic trioxide, actinomycin D, docetaxel(Taxotere), vinblastine, vincristine, nocodazole, colchicines, and othermicrotubule-interfering agents.

Although it is believed the encoded polypeptide may share at least somebiological activities with phosphatase proteins (particularly dualspecificity proteins), a number of methods of determining the exactbiological function of this clone are either known in the art or aredescribed elsewhere herein. Briefly, the function of this clone may bedetermined by applying microarray methodology. Nucleic acidscorresponding to the human phosphatase polynucleotides, in addition to,other clones of the present invention, may be arrayed on microchips forexpression profiling. Depending on which polynucleotide probe is used tohybridize to the slides, a change in expression of a specific gene mayprovide additional insight into the function of this gene based upon theconditions being studied. For example, an observed increase or decreasein expression levels when the polynucleotide probe used comes fromdiseased testis tissue, as compared to, normal tissue might indicate afunction in modulating testis function, for example. In the case ofhuman RET31 phosphatase, adrenal gland, testis, prostate, ovary,skeletal muscle, liver, placenta, pancreas, thymus, small intestine,thyroid, heart, kidney, and/or lung tissue should be used, for example,to extract RNA to prepare the probe.

In addition, the function of the protein may be assessed by applyingquantitative PCR methodology, for example. Real time quantitative PCRwould provide the capability of following the expression of the humanphosphatase gene throughout development, for example. Quantitative PCRmethodology requires only a nominal amount of tissue from eachdevelopmentally important step is needed to perform such experiments.Therefore, the application of quantitative PCR methodology to refiningthe biological function of this polypeptide is encompassed by thepresent invention. In the case of human phosphatase, a diseasecorrelation related to human phosphatase may be made by comparing themRNA expression level of human phosphatase in normal tissue, as comparedto diseased tissue (particularly diseased tissue isolated from thefollowing: adrenal gland, testis, prostate, ovary, skeletal muscle,liver, placenta, pancreas, thymus, small intestine, thyroid, heart,kidney, and/or lung tissue). Significantly higher or lower levels ofhuman phosphatase expression in the diseased tissue may suggest humanphosphatase plays a role in disease progression, and antagonists againsthuman phosphatase polypeptides would be useful therapeutically intreating, preventing, and/or ameliorating the disease. Alternatively,significantly higher or lower levels of human phosphatase expression inthe diseased tissue may suggest human phosphatase plays a defensive roleagainst disease progression, and agonists of human phosphatasepolypeptides may be useful therapeutically in treating, preventing,and/or ameliorating the disease. Also encompassed by the presentinvention are quantitative PCR probes corresponding to thepolynucleotide sequence provided as SEQ ID NO:108 (FIGS. 13A-F).

The function of the protein may also be assessed through complementationassays in yeast. For example, in the case of the human phosphatase,transforming yeast deficient in dual-specificity phosphatase activity,for example, and assessing their ability to grow would provideconvincing evidence the human phosphatase polypeptide hasdual-specificity phosphatase activity. Additional assay conditions andmethods that may be used in assessing the function of thepolynucleotides and polypeptides of the present invention are known inthe art, some of which are disclosed elsewhere herein.

Alternatively, the biological function of the encoded polypeptide may bedetermined by disrupting a homologue of this polypeptide in Mice and/orrats and observing the resulting phenotype. Such knock-out experimentsare known in the art, some of which are disclosed elsewhere herein.

Moreover, the biological function of this polypeptide may be determinedby the application of antisense and/or sense methodology and theresulting generation of transgenic mice and/or rats. Expressing aparticular gene in either sense or antisense orientation in a transgenicmouse or rat could lead to respectively higher or lower expressionlevels of that particular gene. Altering the endogenous expressionlevels of a gene can lead to the observation of a particular phenotypethat can then be used to derive indications on the function of the gene.The gene can be either over-expressed or under expressed in every cellof the organism at all times using a strong ubiquitous promoter, or itcould be expressed in one or more discrete parts of the organism using awell characterized tissue-specific promoter (e.g., a adrenal gland,testis, prostate, ovary, skeletal muscle, liver, placenta, pancreas,thymus, small intestine, thyroid, heart, kidney, and/or lung tissuespecific promoter), or it can be expressed at a specified time ofdevelopment using an inducible and/or a developmentally regulatedpromoter.

In the case of human phosphatase transgenic mice or rats, if nophenotype is apparent in normal growth conditions, observing theorganism under diseased conditions (metabolic, reproductive, immune,hematopoietic, cardiovascular, hepatic, or pulmonary disorders, inaddition to cancers, etc.) may lead to understanding the function of thegene. Therefore, the application of antisense and/or sense methodologyto the creation of transgenic mice or rats to refine the biologicalfunction of the polypeptide is encompassed by the present invention.

In preferred embodiments, the following N-terminal RET31 deletionpolypeptides are encompassed by the present invention: M1-S665, A2-S665,H3-S665, E4-S665, M5-S665, I6-S665, G7-S665, T8-S665, Q9-S665, I10-S665,V11-S665, T12-S665, E13-S665, R14-S665, L15-S665, V16-S665, A17-S665,L18-S665, L19-S665, E20-S665, S21-S665, G22-S665, T23-S665, E24-S665,K25-S665, V26-S665, L27-S665, L28-S665, I29-S665, D30-S665, S31-S665,R32-S665, P33-S665, F34-S665, V35-S665, E36-S665, Y37-S665, N38-S665,T39-S665, S40-S665, H41-S665, I42-S665, L43-S665, E44-S665, A45-S665,I46-S665, N47-S665, I48-S665, N49-S665, C50-S665, S51-S665, K52-S665,L53-S665, M54-S665, K55-S665, R56-S665, R57-S665, L58-S665, Q59-S665,Q60-S665, D61-S665, K62-S665, V63-S665, L64-S665, I65-S665, T66-S665,E67-S665, L68-S665, I69-S665, Q70-S665, H71-S665, S72-S665, A73-S665,K74-S665, H75-S665, K76-S665, V77-S665, D78-S665, I79-S665, D80-S665,C81-S665, S82-S665, Q83-S665, K84-S665, V85-S665, V86-S665, V87-S665,Y88-S665, D89-S665, Q90-S665, S91-S665, S92-S665, Q93-S665, D94-S665,V95-S665, A96-S665, S97-S665, L98-S665, S99-S665, S100-S665, D101-S665,C102-S665, F103-S665, L104-S665, T105-S665, V106-S665, L107-S665,L108-S665, G109-S665, K110-S665, L111-S665, E112-S665, K113-S665,S114-S665, F115-S665, N116-S665, S117-S665, V118-S665, H119-S665,L120-S665, L121-S665, A122-S665, G123-S665, G124-S665, F125-S665,A126-S665, E127-S665, F128-S665, S129-S665, R130-S665, C131-S665,F132-S665, P133-S665, G134-S665, L135-S665, C136-S665, E137-S665,G138-S665, K139-S665, S140-S665, T141-S665, L142-S665, V143-S665,P144-S665, T145-S665, C146-S665, I147-S665, S148-S665, Q149-S665,P150-S665, C151-S665, L152-S665, P153-S665, V154-S665, A155-S665,N156-S665, I157-S665, G158-S665, P159-S665, T160-S665, R161-S665,I162-S665, L163-S665, P164-S665, N165-S665, L166-S665, Y167-S665,L168-S665, G169-S665, C170-S665, Q171-S665, R172-S665, D173-S665,V174-S665, L175-S665, N176-S665, K177-S665, E178-S665, L179-S665,I180-S665, Q181-S665, Q182-S665, N183-S665, G184-S665, I185-S665,G186-S665, Y187-S665, V188-S665, L189-S665, N190-S665, A191-S665,S192-S665, Y193-S665, T194-S665, C195-S665, P196-S665, K197-S665,P198-S665, D199-S665, F200-S665, I201-S665, P202-S665, E203-S665,S204-S665, H205-S665, F206-S665, L207-S665, R208-S665, V209-S665,P210-S665, V21-S665, N212-S665, D213-S665, S214-S665, F215-S665,C216-S665, E217-S665, K218-S665, I219-S665, L220-S665, P221-S665,W222-S665, L223-S665, D224-S665, K225-S665, S226-S665, V227-S665,D228-S665, F229-S665, I230-S665, E231-S665, K232-S665, A233-S665,K234-S665, A235-S665, S236-S665, N237-S665, G238-S665, C239-S665,V240-S665, L241-S665, V242-S665, H243-S665, C244-S665, L245-S665,A246-S665, G247-S665, I248-S665, S249-S665, R250-S665, S251-S665,A252-S665, T253-S665, I254-S665, A255-S665, I256-S665, A257-S665,Y258-S665, I259-S665, M260-S665, K261-S665, R262-S665, M263-S665,D264-S665, M265-S665, S266-S665, L267-S665, D268-S665, E269-S665,A270-S665, Y271-S665, R272-S665, F273-S665, V274-S665, K275-S665,E276-S665, K277-S665, R278-S665, P279-S665, T280-S665, I281-S665,S282-S665, P283-S665, N284-S665, F285-S665, N286-S665, F287-S665,L288-S665, G289-S665, Q290-S665, L291-S665, L292-S665, D293-S665,Y294-S665, E295-S665, K296-S665, K297-S665, I298-S665, K299-S665,N300-S665, Q301-S665, T302-S665, G303-S665, A304-S665, S305-S665,G306-S665, P307-S665, K308-S665, S309-S665, K310-S665, L311-S665,K312-S665, L313-S665, L314-S665, H315-S665, L316-S665, E317-S665,K318-S665, P319-S665, N320-S665, E321-S665, P322-S665, V323-S665,P324-S665, A325-S665, V326-S665, S327-S665, E328-S665, G329-S665,G330-S665, Q331-S665, K332-S665, S333-S665, E334-S665, T335-S665,P336-S665, L337-S665, S338-S665, P339-S665, P340-S665, C341-S665,A342-S665, D343-S665, S344-S665, A345-S665, T346-S665, S347-S665,E348-S665, A349-S665, A350-S665, G351-S665, Q352-S665, R353-S665,P354-S665, V355-S665, H356-S665, P357-S665, A358-S665, S359-S665,V360-S665, P361-S665, S362-S665, V363-S665, P364-S665, S365-S665,V366-S665, Q367-S665, P368-S665, S369-S665, L370-S665, L371-S665,E372-S665, D373-S665, S374-S665, P375-S665, L376-S665, V377-S665,Q378-S665, A379-S665, L380-S665, S381-S665, G382-S665, L383-S665,H384-S665, L385-S665, S386-S665, A387-S665, D388-S665, R389-S665,L390-S665, E391-S665, D392-S665, S393-S665, N394-S665, K395-S665,L396-S665, K397-S665, R398-S665, S399-S665, F400-S665, S401-S665,L402-S665, D403-S665, I404-S665, K405-S665, S406-S665, V407-S665,S408-S665, Y409-S665, S410-S665, A411-S665, S412-S665, M413-S665,A414-S665, A415-S665, S416-S665, L417-S665, H418-S665, G419-S665,F420-S665, S421-S665, S422-S665, S423-S665, E424-S665, D425-S665,A426-S665, L427-S665, E428-S665, Y429-S665, Y430-S665, K431-S665,P432-S665, S433-S665, T434-S665, T435-S665, L436-S665, D437-S665,G438-S665, T439-S665, N440-S665, K441-S665, L442-S665, C443-S665,Q444-S665, F445-S665, S446-S665, P447-S665, V448-S665, Q449-S665,E450-S665, L451-S665, S452-S665, E453-S665, Q454-S665, T455-S665,P456-S665, E457-S665, T458-S665, S459-S665, P460-S665, D461-S665,K462-S665, E463-S665, E464-S665, A465-S665, S466-S665, I467-S665,P468-S665, K469-S665, K470-S665, L471-S665, Q472-S665, T473-S665,A474-S665, R475-S665, P476-S665, S477-S665, D478-S665, S479-S665,Q480-S665, S481-S665, K482-S665, R483-S665, L484-S665, H485-S665,S486-S665, V487-S665, R488-S665, T489-S665, S490-S665, S491-S665,S492-S665, G493-S665, T494-S665, A495-S665, Q496-S665, R497-S665,S498-S665, L499-S665, L500-S665, S501-S665, P502-S665, L503-S665,H504-S665, R505-S665, S506-S665, G507-S665, S508-S665, V509-S665,E510-S665, D51-S665, N512-S665, Y513-S665, H514-S665, T515-S665,S516-S665, F517-S665, L518-S665, F519-S665, G520-S665, L521-S665,S522-S665, T523-S665, S524-S665, Q525-S665, Q526-S665, H527-S665,L528-S665, T529-S665, K530-S665, S531-S665, A532-S665, G533-S665,L534-S665, G535-S665, L536-S665, K537-S665, G538-S665, W539-S665,H540-S665, S541-S665, D542-S665, I543-S665, L544-S665, A545-S665,P546-S665, Q547-S665, T548-S665, S549-S665, T550-S665, P551-S665,S552-S665, L553-S665, T554-S665, S555-S665, S556-S665, W557-S665,Y558-S665, F559-S665, A560-S665, T561-S665, E562-S665, S563-S665,S564-S665, H565-S665, F566-S665, Y567-S665, S568-S665, A569-S665,S570-S665, A571-S665, I572-S665, Y573-S665, G574-S665, G575-S665,S576-S665, A577-S665, S578-S665, Y579-S665, S580-S665, A581-S665,Y582-S665, S583-S665, C584-S665, S585-S665, Q586-S665, L587-S665,P588-S665, T589-S665, C590-S665, G591-S665, D592-S665, Q593-S665,V594-S665-Y595-S665, S596-S665, V597-S665, R598-S665, R599-S665,R600-S665, Q601-S665, K602-S665, P603-S665, S604-S665, D605-S665,R606-S665, A607-S665, D608-S665, S609-S665, R610-S665, R611-S665,S612-S665, W613-S665, H614-S665, E615-S665, E616-S665, S617S665,P618-S665, F619-S665, E620-S665, K621-S665, Q622-S665, F623-S665,K624-S665, R625-S665, R626-S665, S627-S665, C628-S665, Q629-S665,M630-S665, E631-S665, F632-S665, G633-S665, E634-S665, S635-S665,I636-S665, M637-S665, S638-S665, E639-S665, N640-S665, R641-S665,S642-S665, R643-S665, E644-S665, E645-S665, L646-S665, G647-S665,K648-S665, V649-S665, G650-S665, S651-S665, Q652-S665, S653-S665,S654-S665, F655-S665, S656-S665, G657-S665, S658-S665, and/or M659-S665of SEQ ID NO:109. Polynucleotide sequences encoding these polypeptidesare also provided. The present invention also encompasses the use ofthese N-terminal RET31 deletion polypeptides as immunogenic and/orantigenic epitopes as described elsewhere herein.

In preferred embodiments, the following C-terminal RET31 deletionpolypeptides are encompassed by the present invention: M1-S665, M1-V664,M1-E663, M1-I662, M1-I661, M1-E660, M1-M659, M1-S658, M1-G657, M1-S656,M1-F655, M1-S654, M1-S653, M1-Q652, M1-S651, M1-G650, M1-V649, M1-K648,M1-G647, M1-L646, M1-E645, M1-E644, M1-R643, M1-S642, M1-R641, M1-N640,M1-E639, M1-S638, M1-M637, M1-I636, M1-S635, M1-E634, M1-G633, M1-F632,M1-E631, M1-M630, M1-Q629, M1-C628, M1-S627, M1-R626, M1-R625, M1-K624,M1-F623, M1-Q622, M1-K621, M1-E620, M1-F619, M1-P618, M1-S617, M1-E616,M1-E615, M1-H614, M1-W613, M1-S612, M1-R611, M1-R610, M1-S609, M1-D608,M1-A607, M1-R606, M1-D605, M1-S604, M1-P603, M1-K602, M1-Q601, M1-R600,M1-R599, M1-R598, M1-V597, M1-S596, M1-Y595, M1-S594, M1-Q593, M1-D592,M1-G591, M1-C590, M1-T589, M1-P588, M1-L587, M1-Q586, M1-S585, M1-C584,M1-S583, M1-Y582, M1-A581, M1-S580, M1-Y579, M1-S578, M1-A577, M1-S576,M1-G575, M1-G574, M1-Y573, M1-I572, M1-A571, M1-S570, M1-A569, M1-S568,M1-Y567, M1-F566, M1-H565, M1-S564, M1-S563, M1-E562, M1-T561, M1-A560,M1-F559, M1-Y558, M1-W557, M1-S556, M1-S555, M1-T554, M1-L553, M1-S552,M1-P551, M1-T550, M1-S549, M1-T548, M1-Q547, M1-P546, M1-A545, M1-L544,M1-I543, M1-D542, M1-S541, M1-H540, M1-W539, M1-G538, M1-K537, M1-L536,M1-G535, M1-L534, M1-G533, M1-A532, M1-S531, M1-K530, M1-T529, M1-L528,M1-H527, M1-Q526, M1-Q525, M1-S524, M1-T523, M1-S522, M1-L521, M1-G520,M1-F519, M1-L518, M1-F517, M1-S516, M1-T515, M1-H514, M1-Y513, M1-N512,M1-D511, M1-E510, M1-V509, M1-S508, M1-G507, M1-S506, M1-R505, M1-H504,M1-L503, M1-P502, M1-S501, M1-L500, M1-L499, M1-S498, M1-R497, M1-Q496,M1-A495, M1-T494, M1-G493, M1-S492, M1-S491, M1-S490, M1-T489, M1-R488,M1-V487, M1-S486, M1-H485, M1-L484, M1-R483, M1-K482, M1-S481, M1-Q480,M1-S479, M1-D478, M1-S477, M1-P476, M1-R475, M1-A474, M1-T473, M1-Q472,M1-L471, M1-K470, M1-K469, M1-P468, M1-I467, M1-S466, M1-A465, M1-E464,M1-E463, M1-K462, M1-D461, M1-P460, M1-S459, M1-T458, M1-E457, M1-P456,M1-T455, M1-Q454, M1-E453, M1-S452, M1-L451, M1-E450, M1-Q449, M1-V448,M1-P447, M1-S446, M1-F445, M1-Q444, M1-C443, M1-L442, M1-K441, M1-N440,M1-T439, M1-G438, M1-D437, M1-L436, M1-T435, M1-T434, M1-S433, M1-P432,M1-K431, M1-Y430, M1-Y429, M1-E428, M1-L427, M1-A426, M1-D425, M1-E424,M1-S423, M1-S422, M1-S421, M1-F420, M1-G419, M1-H418, M1-L417, M1-S416,M1-A415, M1-A414, M1-M413, M1-S412, M1-A411, M1-S410, M1-Y409, M1-S408,M1-V407, M1-S406, M1-K405, M1-I404, M1-D403, M1-L402, M1-S401, M1-F400,M1-S399, M1-R398, M1-K397, M1-L396, M1-K395, M1-N394, M1-S393, M1-D392,M1-E391, M1-L390, M1-R389, M1-D388, M1-A387, M1-S386, M1-L385, M1-H384,M1-L383, M1-G382, M1-S381, M1-L380, M1-A379, M1-Q378, M1-V377, M1-L376,M1-P375, M1-S374, M1-D373, M1-E372, M1-L371, M1-L370, M1-S369, M1-P368,M1-Q367, M1-V366, M1-S365, M1-P364, M1-V363, M1-S362, M1-P361, M1-V360,M1-S359, M1-A358, M1-P357, M1-H356, M1-V355, M1-P354, M1-R353, M1-Q352,M1-G351, M1-A350, M1-A349, M1-E348, M1-S347, M1-T346, M1-A345, M1-S344,M1-D343, M1-A342, M1-C341, M1-P340, M1-P339, M1-S338, M1-L337, M1-P336,M1-T335, M1-E334, M1-S333, M1-K332, M1-Q331, M1-G330, M1-G329, M1-E328,M1-S327, M1-V326, M1-A325, M1-P324, M1-V323, M1-P322, M1-E321, M1-N320,M1-P319, M1-K318, M1-E317, M1-L316, M1-H315, M1-L314, M1-L313, M1-K312,M1-L311, M1-K310, M1-S309, M1-K308, M1-P307, M1-G306, M1-S305, M1-A304,M1-G303, M1-T302, M1-Q301, M1-N300, M1-K299, M1-I298, M1-K297, M1-K296,M1-E295, M1-Y294, M1-D293, M1-L292, M1-L291, M1-Q290, M1-G289, M1-L288,M1-F287, M1-N286, M1-F285, M1-N284, M1-P283, M1-S282, M1-I281, M1-T280,M1-P279, M1-R278, M1-K277, M1-E276, M1-K275, M1-V274, M1-F273, M1-R272,M1-Y271, M1-A270, M1-E269, M1-D268, M1-L267, M1-S266, M1-M265, M1-D264,M1-M263, M1-R262, M1-K261, M1-M260, M1-I259, M1-Y258, M1-A257, M1-I256,M1-A255, M1-I254, M1-T253, M1-A252, M1-S251, M1-R250, M1-S249, M1-I248,M1-G247, M1-A246, M1-L245, M1-C244, M1-H243, M1-V242, M1-L241, M1-V240,M1-C239, M1-G238, M1-N237, M1-S236, M1-A235, M1-K234, M1-A233, M1-K232,M1-E231, M1-I230, M1-F229, M1-D228, M1-V227, M1-S226, M1-K225, M1-D224,M1-L223, M1-W222, M1-P221, M1-L220, M1-I219, M1-K218, M1-E217, M1-C216,M1-F215, M1-S214, M1-D213, M1-N212, M1-V211, M1-P210, M1-V209, M1-R208,M1-L207, M1-F206, M1-H205, M1-S204, M1-E203, M1-P202, M1-I201, M1-F200,M1-D199, M1-P198, M1-K197, M1-P196, M1-C195, M1-T194, M1-Y193, M1-S192,M1-A191, M1-N190, M1-L189, M1-V188, M1-Y187, M1-G186, M1-I185, M1-G184,M1-N183, M1-Q182, M1-Q181, M1-I180, M1-L179, M1-E178, M1-K177, M1-N176,M1-L175, M1-V174, M1-D173, M1-R172, M1-Q171, M1-C170, M1-G169, M1-L168,M1-Y167, M1-L166, M1-N165, M1-P164, M1-L163, M1-I162, M1-R161, M1-T160,M1-P159, M1-G158, M1-I157, M1-N156, M1-A155, M1-V154, M1-P153, M1-L152,M1-C151, M1-P150, M1-Q149, M1-S148, M1-I147, M1-C146, M1-T145, M1-P144,M1-V143, M1-L142, M1-T141, M1-S140, M1-K139, M1-G138, M1-E137, M1-C136,M1-L135, M1-G134, M1-P133, M1-F132, M1-C131, M1-R130, M1-S129, M1-F128,M1-E127, M1-A126, M1-F125, M1-G124, M1-G123, M1-A122, M1-L121, M1-L120,M1-H119, M1-V118, M1-S117, M1-N116, M1-F115, M1-S114, M1-K113, M1-E112,M1-L111, M1-K110, M1-G109, M1-L108, M1-L107, M1-V106, M1-T105, M1-L104,M1-F103, M1-C102, M1-D101, M1-S100, M1-S99, M1-L98, M1-S97, M1-A96,M1-V95, M1-D94, M1-Q93, M1-S92, M1-S91, M1-Q90, M1-D89, M1-Y88, M1-V87,M1-V86, M1-V85, M1-K84, M1-Q83, M1-S82, M1-C81, M1-D80, M1-I79, M1-D78,M1-V77, M1-K76, M1-H75, M1-K74, M1-A73, M1-S72, M1-H71, M1-Q70, M1-I69,M1-L68, M1-E67, M1-T66, M1-I65, M1-L64, M1-V63, M1-K62, M1-D61, M1-Q60,M1-Q59, M1-L58, M1-R57, M1-R56, M1-K55, M1-M54, M1-L53, M1-K52, M1-S51,M1-C50, M1-N49, M1-I48, M1-N47, M1-I46, M1-A45, M1-E44, M1-L43, M1-I42,M1-H41, M1-S40, M1-T39, M1-N38, M1-Y37, M1-E36, M1-V35, M1-F34, M1-P33,M1-R32, M1-S31, M1-D30, M1-I29, M1-L28, M1-L27, M1-V26, M1-K25, M1-E24,M1-T23, M1-G22, M1-S21, M1-E20, M1-L19, M1-L18, M1-A17, M1-V16, M1-L15,M1-R14, M1-E13, M1-T12, M1-V11, M1-I10, M1-Q9, M1-T8, and/or M 1-G7 ofSEQ ID NO:109. Polynucleotide sequences encoding these polypeptides arealso provided. The present invention also encompasses the use of theseC-terminal RET31 deletion polypeptides as immunogenic and/or antigenicepitopes as described elsewhere herein.

The present invention also encompasses immunogenic and/or antigenicepitopes of the human RET31 phosphatase polypeptide.

The human phosphatase polypeptides of the present invention weredetermined to comprise several phosphorylation sites based upon theMotif algorithm (Genetics Computer Group, Inc.). The phosphorylation ofsuch sites may regulate some biological activity of the humanphosphatase polypeptide. For example, phosphorylation at specific sitesmay be involved in regulating the proteins ability to associate or bindto other molecules (e.g., proteins, ligands, substrates, DNA, etc.). Inthe present case, phosphorylation may modulate the ability of the humanphosphatase polypeptide to associate with other polypeptides,particularly cognate ligand for human phosphatase, or its ability tomodulate certain cellular signal pathways.

The human phosphatase polypeptide was predicted to comprise twelve PKCphosphorylation sites using the Motif algorithm (Genetics ComputerGroup, Inc.). In vivo, protein kinase C exhibits a preference for thephosphorylation of serine or threonine residues. The PKC phosphorylationsites have the following consensus pattern: [ST]-x-[RK], where S or Trepresents the site of phosphorylation and ‘x’ an intervening amino acidresidue. Additional information regarding PKC phosphorylation sites canbe found in Woodget J. R., Gould K. L., Hunter T., Eur. J. Biochem.161:177-184 (1986), and Kishimoto A., Nishiyama K., Nakanishi H.,Uratsuji Y., Nomura H., Takeyama Y., Nishizuka Y., J. Biol. Chem.260:12492-12499 (1985); which are hereby incorporated by referenceherein.

In preferred embodiments, the following PKC phosphorylation sitepolypeptides are encompassed by the present invention: GTQIVTERLVALL(SEQ ID NO:116), LLESGTEKVLLID (SEQ ID NO:117), ELIQHSAKHKVDI (SEQ IDNO:118), VDIDCSQKVVVYD (SEQ ID NO:119), DRLEDSNKLKRSF (SEQ ID NO:120),TTLDGTNKLCQFS (SEQ ID NO:121), PKKLQTARPSDSQ (SEQ ID NO:122),PSDSQSKRLHSVR (SEQ ID NO:123), SKRLHSVRTSSSG (SEQ ID NO:124),GDQVYSVRRRQKP (SEQ ID NO:125), RRQKPSDRADSRR (SEQ ID NO:126), and/orSDRADSRRSWHEE (SEQ ID NO:127). Polynucleotides encoding thesepolypeptides are also provided. The present invention also encompassesthe use of the human RET31 phosphatase PKC phosphorylation sitepolypeptides as immunogenic and/or antigenic epitopes as describedelsewhere herein.

The human phosphatase polypeptide has been shown to comprise sixglycosylation sites according to the Motif algorithm (Genetics ComputerGroup, Inc.). As discussed more specifically herein, proteinglycosylation is thought to serve a variety of functions including:augmentation of protein folding, inhibition of protein aggregation,regulation of intracellular trafficking to organelles, increasingresistance to proteolysis, modulation of protein antigenicity, andmediation of intercellular adhesion.

Asparagine phosphorylation sites have the following consensus pattern,N-{P}-[ST]-{P}, wherein N represents the glycosylation site. However, itis well known that that potential N-glycosylation sites are specific tothe consensus sequence Asn-Xaa-Ser/Thr. However, the presence of theconsensus tripeptide is not sufficient to conclude that an asparagineresidue is glycosylated, due to the fact that the folding of the proteinplays an important role in the regulation of N-glycosylation. It hasbeen shown that the presence of proline between Asn and Ser/Thr willinhibit N-glycosylation; this has been confirmed by a recent statisticalanalysis of glycosylation sites, which also shows that about 50% of thesites that have a proline C-terminal to Ser/Thr are not glycosylated.Additional information relating to asparagine glycosylation may be foundin reference to the following publications, which are herebyincorporated by reference herein: Marshall R. D., Annu. Rev. Biochem.41:673-702 (1972); Pless D. D., Lennarz W. J., Proc. Natl. Acad. Sci.U.S.A. 74:134-138 (1977); Bause E., Biochem. J. 209:331-336 (1983);Gavel Y., von Heijne G., Protein Eng. 3:433442 (1990); and Miletich J.P., Broze G. J. Jr., J. Biol. Chem. 265:11397-11404 (1990).

In preferred embodiments, the following asparagine glycosylation sitepolypeptides are encompassed by the present invention: PFVEYNTSHILEAI(SEQ ID NO:128), EAININCSKLMKRR (SEQ ID NO:129), IGYVLNASYTCPKP (SEQ IDNO:130), LRVPVNDSFCEKIL (SEQ ID NO:131), EKKIKNQTGASGPK (SEQ ID NO:132),and/or SIMSENRSREELGK (SEQ ID NO:133). Polynucleotides encoding thesepolypeptides are also provided. The present invention also encompassesthe use of the human RET31 phosphatase asparagine glycosylation sitepolypeptides as immunogenic and/or antigenic epitopes as describedelsewhere herein.

In confirmation of the human RET31 representing a novel humanphosphatase polypeptide, the RET31 polypeptide has been shown tocomprise a dual specificity phosphatase catalytic domain as identifiedby the BLAST2 algorithm using the DSPc PFAM HMM (PF00782) as a querysequence.

The catalytic residue of the human RET31 polypeptide is represented byan acitve site cysteine located at amino acid residue 244 of SEQ IDNO:109 (FIGS. 13A-F).

In preferred embodiments, the following human RET31 DSPc domainpolypeptide is encompassed by the present invention:GPTRLPNLYLGCQRDVLNKELIQQNGIGYVLNASYTCPKPDFIPESHFLRVPVNDSFCEKILPWLDKSVDFIEKAKASNGCVLVHCLAGISRSATIAIAYIMKRMDMSLDEAYRFVKEKRPTISPNFNFLGQLLDYEKK (SEQ ID NO:134). Polynucleotides encoding thispolypeptide are also provided. The present invention also encompassesthe use of this human RET31 DSPc domain polypeptide as an immunogenicand/or antigenic epitope as described elsewhere herein.

In preferred embodiments, the following human RET31 DSPc domain aminoacid substitutions are encompassed by the present invention: whereinG158 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P,Q, R, S, T, V, W, or Y; wherein P159 is substituted with either an A, C,D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein T160 issubstituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, V, W, or Y; wherein R161 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein I162 is substitutedwith either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, orY; wherein L163 is substituted with either an A, C, D, E, F, G, H, I, K,M, N, P, Q, R, S, T, V, W, or Y; wherein P164 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; whereinN165 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P,Q, R, S, T, V, W, or Y; wherein L166 is substituted with either an A, C,D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein Y167 issubstituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, T, V, or W; wherein L168 is substituted with either an A, C, D, E, F,G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein G169 is substitutedwith either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, orY; wherein C170 is substituted with either an A, D, E, F, G, H, I, K, L,M, N, P, Q, R, S, T, V, W, or Y; wherein Q171 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; whereinR172 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N,P, Q, S, T, V, W, or Y; wherein D173 is substituted with either an A, C,E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V174 issubstituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, T, W, or Y; wherein L175 is substituted with either an A, C, D, E, F,G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein N176 is substitutedwith either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, orY; wherein K177 is substituted with either an A, C, D, E, F, G, H, I, L,M, N, P, Q, R, S, T, V, W, or Y; wherein E178 is substituted with eitheran A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; whereinL179 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P,Q, R, S, T, V, W, or Y; wherein I180 is substituted with either an A, C,D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Q181 issubstituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S,T, V, W, or Y; wherein Q182 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein N183 is substitutedwith either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, orY; wherein G184 is substituted with either an A, C, D, E, F, H, I, K, L,M, N, P, Q, R, S, T, V, W, or Y; wherein I185 is substituted with eitheran A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; whereinG186 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P,Q, R, S, T, V, W, or Y; wherein Y187 is substituted with either an A, C,D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein V188 issubstituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, T, W, or Y; wherein L189 is substituted with either an A, C, D, E, F,G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein N190 is substitutedwith either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, orY; wherein A191 is substituted with either a C, D, E, F, G, H, I, K, L,M, N, P, Q, R, S, T, V, W, or Y; wherein S192 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; whereinY193 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N,P, Q, R, S, T, V, or W; wherein T194 is substituted with either an A, C,D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein C195 issubstituted with either an A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,T, V, W, or Y; wherein P196 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein K197 is substitutedwith either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, orY; wherein P198 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, N, Q, R, S, T, V, W, or Y; wherein D199 is substituted with eitheran A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; whereinF200 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P,Q, R, S, T, V, W, or Y; wherein I201 is substituted with either an A, C,D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein P202 issubstituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S,T, V, W, or Y; wherein E203 is substituted with either an A, C, D, F, G,H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein S204 is substitutedwith either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, orY; wherein H205 is substituted with either an A, C, D, E, F, G, I, K, L,M, N, P, Q, R, S, T, V, W, or Y; wherein F206 is substituted with eitheran A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; whereinL207 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P,Q, R, S, T, V, W, or Y; wherein R208 is substituted with either an A, C,D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein V209 issubstituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, T, W, or Y; wherein P210 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein V211 is substitutedwith either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, orY; wherein N212 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, P, Q, R, S, T, V, W, or Y; wherein D213 is substituted with eitheran A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; whereinS214 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N,P, Q, R, T, V, W, or Y; wherein F215 is substituted with either an A, C,D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein C216 issubstituted with either an A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,T, V, W, or Y; wherein E217 is substituted with either an A, C, D, F, G,H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein K218 is substitutedwith either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, orY; wherein I219 is substituted with either an A, C, D, E, F, G, H, K, L,M, N, P, Q, R, S, T, V, W, or Y; wherein L220 is substituted with eitheran A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; whereinP221 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N,Q, R, S, T, V, W, or Y; wherein W222 is substituted with either an A, C,D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein L223 issubstituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S,T, V, W, or Y; wherein D224 is substituted with either an A, C, E, F, G,H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein K225 is substitutedwith either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, orY; wherein S226 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, N, P, Q, R, T, V, W, or Y; wherein V227 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; whereinD228 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P,Q, R, S, T, V, W, or Y; wherein F229 is substituted with either an A, C,D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein I230 issubstituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S,T, V, W, or Y; wherein E231 is substituted with either an A, C, D, F, G,H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein K232 is substitutedwith either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, orY; wherein A233 is substituted with either a C, D, E, F, G, H, I, K, L,M, N, P, Q, R, S, T, V, W, or Y; wherein K234 is substituted with eitheran A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; whereinA235 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q,R, S, T, V, W, or Y; wherein S236 is substituted with either an A, C, D,E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein N237 issubstituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S,T, V, W, or Y; wherein G238 is substituted with either an A, C, D, E, F,H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein C239 is substitutedwith either an A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, orY; wherein V240 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, N, P, Q, R, S, T, W, or Y; wherein L241 is substituted with eitheran A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; whereinV242 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N,P, Q, R, S, T, W, or Y; wherein H243 is substituted with either an A, C,D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein C244 issubstituted with either an A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,T, V, W, or Y; wherein L245 is substituted with either an A, C, D, E, F,G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein A246 is substitutedwith either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, orY; wherein G247 is substituted with either an A, C, D, E, F, H, I, K, L,M, N, P, Q, R, S, T, V, W, or Y; wherein I248 is substituted with eitheran A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; whereinS249 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N,P, Q, R, T, V, W, or Y; wherein R250 is substituted with either an A, C,D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein S251 issubstituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,T, V, W, or Y; wherein A252 is substituted with either a C, D, E, F, G,H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein T253 is substitutedwith either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, orY; wherein I254 is substituted with either an A, C, D, E, F, G, H, K, L,M, N, P, Q, R, S, T, V, W, or Y; wherein A255 is substituted with eithera C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; whereinI256 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P,Q, R, S, T, V, W, or Y; wherein A257 is substituted with either a C, D,E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Y258 issubstituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, T, V, or W; wherein I259 is substituted with either an A, C, D, E, F,G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein M260 is substitutedwith either an A, C, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, orY; wherein K261 is substituted with either an A, C, D, E, F, G, H, I, L,M, N, P, Q, R, S, T, V, W, or Y; wherein R262 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; whereinM263 is substituted with either an A, C, D, E, F, G, H, I, K, L, N, P,Q, R, S, T, V, W, or Y; wherein D264 is substituted with either an A, C,E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein M265 issubstituted with either an A, C, D, E, F, G, H, I, K, L, N, P, Q, R, S,T, V, W, or Y; wherein S266 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein L267 is substitutedwith either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, orY; wherein D268 is substituted with either an A, C, E, F, G, H, I, K, L,M, N, P, Q, R, S, T, V, W, or Y; wherein E269 is substituted with eitheran A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; whereinA270 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q,R, S, T, V, W, or Y; wherein Y271 is substituted with either an A, C, D,E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein R272 issubstituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S,T, V, W, or Y; wherein F273 is substituted with either an A, C, D, E, G,H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V274 is substitutedwith either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, orY; wherein K275 is substituted with either an A, C, D, E, F, G, H, I, L,M, N, P, Q, R, S, T, V, W, or Y; wherein E276 is substituted with eitheran A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; whereinK277 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P,Q, R, S, T, V, W, or Y; wherein R278 is substituted with either an A, C,D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein P279 issubstituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S,T, V, W, or Y; wherein T280 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein I281 is substitutedwith either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, orY; wherein S282 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, N, P, Q, R, T, V, W, or Y; wherein P283 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; whereinN284 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P,Q, R, S, T, V, W, or Y; wherein F285 is substituted with either an A, C,D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein N286 issubstituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S,T, V, W, or Y; wherein F287 is substituted with either an A, C, D, E, G,H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L288 is substitutedwith either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, orY; wherein G289 is substituted with either an A, C, D, E, F, H, I, K, L,M, N, P, Q, R, S, T, V, W, or Y; wherein Q290 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; whereinL291 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P,Q, R, S, T, V, W, or Y; wherein L292 is substituted with either an A, C,D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein D293 issubstituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S,T, V, W, or Y; wherein Y294 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein E295 is substitutedwith either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, orY; wherein K296 is substituted with either an A, C, D, E, F, G, H, I, L,M, N, P, Q, R, S, T, V, W, or Y; and/or wherein K297 is substituted witheither an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y ofSEQ ID NO:109, in addition to any combination thereof. The presentinvention also encompasses the use of these human RET31 DSPc domainamino acid substituted polypeptides as immunogenic and/or antigenicepitopes as described elsewhere herein.

In preferred embodiments, the following human RET31 DSPc domainconservative amino acid substitutions are encompassed by the presentinvention: wherein G158 is substituted with either an A, M, S, or T;wherein P159 is a P; wherein T160 is substituted with either an A, G, M,or S; wherein R161 is substituted with either a K, or H; wherein I162 issubstituted with either an A, V, or L; wherein L163 is substituted witheither an A, I, or V; wherein P164 is a P; wherein N165 is substitutedwith a Q; wherein L166 is substituted with either an A, I, or V; whereinY167 is either an F, or W; wherein L168 is substituted with either an A,I, or V; wherein G169 is substituted with either an A, M, S, or T;wherein C170 is a C; wherein Q171 is substituted with a N; wherein R172is substituted with either a K, or H; wherein D173 is substituted withan E; wherein V174 is substituted with either an A, I, or L; whereinL175 is substituted with either an A, I, or V; wherein N176 issubstituted with a Q; wherein K177 is substituted with either a R, or H;wherein E178 is substituted with a D; wherein L179 is substituted witheither an A, I, or V; wherein I180 is substituted with either an A, V,or L; wherein Q181 is substituted with a N; wherein Q182 is substitutedwith a N; wherein N183 is substituted with a Q; wherein G184 issubstituted with either an A, M, S, or T; wherein I185 is substitutedwith either an A, V, or L; wherein G186 is substituted with either an A,M, S, or T; wherein Y187 is either an F, or W; wherein V188 issubstituted with either an A, I, or L; wherein L189 is substituted witheither an A, I, or V; wherein N190 is substituted with a Q; wherein A191is substituted with either a G, I, L, M, S, T, or V; wherein S192 issubstituted with either an A, G, M, or T; wherein Y193 is either an F,or W; wherein T194 is substituted with either an A, G, M, or S; whereinC195 is a C; wherein P196 is a P; wherein K197 is substituted witheither a R, or H; wherein P198 is a P; wherein D199 is substituted withan E; wherein F200 is substituted with either a W, or Y; wherein I201 issubstituted with either an A, V, or L; wherein P202 is a P; wherein E203is substituted with a D; wherein S204 is substituted with either an A,G, M, or T; wherein H205 is substituted with either a K, or R; whereinF206 is substituted with either a W, or Y; wherein L207 is substitutedwith either an A, I, or V; wherein R208 is substituted with either a K,or H; wherein V209 is substituted with either an A, I, or L; whereinP210 is a P; wherein V211 is substituted with either an A, I, or L;wherein N212 is substituted with a Q; wherein D213 is substituted withan E; wherein S214 is substituted with either an A, G, M, or T; whereinF215 is substituted with either a W, or Y; wherein C216 is a C; whereinE217 is substituted with a D; wherein K218 is substituted with either aR, or H; wherein I219 is substituted with either an A, V, or L; whereinL220 is substituted with either an A, I, or V; wherein P221 is a P;wherein W222 is either an F, or Y; wherein L223 is substituted witheither an A, I, or V; wherein D224 is substituted with an E; whereinK225 is substituted with either a R, or H; wherein S226 is substitutedwith either an A, G, M, or T; wherein V227 is substituted with either anA, I, or L; wherein D228 is substituted with an E; wherein F229 issubstituted with either a W, or Y; wherein I230 is substituted witheither an A, V, or L; wherein E231 is substituted with a D; wherein K232is substituted with either a R, or H; wherein A233 is substituted witheither a G, I, L, M, S, T, or V; wherein K234 is substituted with eithera R, or H; wherein A235 is substituted with either a G, I, L, M, S, T,or V; wherein S236 is substituted with either an A, G, M, or T; whereinN237 is substituted with a Q; wherein G238 is substituted with either anA, M, S, or T; wherein C239 is a C; wherein V240 is substituted witheither an A, I, or L; wherein L241 is substituted with either an A, I,or V; wherein V242 is substituted with either an A, I, or L; whereinH243 is substituted with either a K, or R; wherein C244 is a C; whereinL245 is substituted with either an A, I, or V; wherein A246 issubstituted with either a G, I, L, M, S, T, or V; wherein G247 issubstituted with either an A, M, S, or T; wherein I248 is substitutedwith either an A, V, or L; wherein S249 is substituted with either an A,G, M, or T; wherein R250 is substituted with either a K, or H; whereinS251 is substituted with either an A, G, M, or T; wherein A252 issubstituted with either a G, I, L, M, S, T, or V; wherein T253 issubstituted with either an A, G, M, or S; wherein I254 is substitutedwith either an A, V, or L; wherein A255 is substituted with either a G,I, L, M, S, T, or V; wherein I256 is substituted with either an A, V, orL; wherein A257 is substituted with either a G, I, L, M, S, T, or V;wherein Y258 is either an F, or W; wherein I259 is substituted witheither an A, V, or L; wherein M260 is substituted with either an A, G,S, or T; wherein K261 is substituted with either a R, or H; wherein R262is substituted with either a K, or H; wherein M263 is substituted witheither an A, G, S, or T; wherein D264 is substituted with an E; whereinM265 is substituted with either an A, G, S, or T; wherein S266 issubstituted with either an A, G, M, or T; wherein L267 is substitutedwith either an A, I, or V; wherein D268 is substituted with an E;wherein E269 is substituted with a D; wherein A270 is substituted witheither a G, I, L, M, S, T, or V; wherein Y271 is either an F, or W;wherein R272 is substituted with either a K, or H; wherein F273 issubstituted with either a W, or Y; wherein V274 is substituted witheither an A, I, or L; wherein K275 is substituted with either a R, or H;wherein E276 is substituted with a D; wherein K277 is substituted witheither a R, or H; wherein R278 is substituted with either a K, or H;wherein P279 is a P; wherein T280 is substituted with either an A, G, M,or S; wherein I281 is substituted with either an A, V, or L; whereinS282 is substituted with either an A, G, M, or T; wherein P283 is a P;wherein N284 is substituted with a Q; wherein F285 is substituted witheither a W, or Y; wherein N286 is substituted with a Q; wherein F287 issubstituted with either a W, or Y; wherein L288 is substituted witheither an A, I, or V; wherein G289 is substituted with either an A, M,S, or T; wherein Q290 is substituted with a N; wherein L291 issubstituted with either an A, I, or V; wherein L292 is substituted witheither an A, I, or V; wherein D293 is substituted with an E; whereinY294 is either an F, or W; wherein E295 is substituted with a D; whereinK296 is substituted with either a R, or H; and/or wherein K297 issubstituted with either a R, or H of SEQ ID NO:109 in addition to anycombination thereof. Other suitable substitutions within the human RET31DSPc domain are encompassed by the present invention and are referencedelsewhere herein. The present invention also encompasses the use ofthese human RET31 DSPc domain conservative amino acid substitutedpolypeptides as immunogenic and/or antigenic epitopes as describedelsewhere herein.

In further confirmation of the human RET31 polypeptide representing anovel human phosphatase polypeptide, the RET31 polypeptide has beenshown to comprise a tyrosine specific protein phosphatase active sitedomain according to the Motif algorithm (Genetics Computer Group, Inc.).

Tyrosine specific protein phosphatases (EC 3.1.3.48) (PTPase) areenzymes that catalyze the removal of a phosphate group attached to atyrosine residue. These enzymes are very important in the control ofcell growth, proliferation, differentiation and transformation. Multipleforms of PTPase have been characterized and can be classified into twocategories: soluble PTPases and transmembrane receptor proteins thatcontain PTPase domain(s).

The currently known PTPases are listed below: Soluble PTPases, PTPN1(PTP-1B), PTPN2 (T-cell PTPase; TC-PTP), PTPN3 (H1) and PTPN4 (MEG),enzymes that contain an N-terminal band 4.1-like domain and could act atjunctions between the membrane and cytoskeleton, PTPN5 (STEP), PTPN6(PTP-1C; HCP; SHP) and PTPN11 (PTP-2C; SH-PTP3; Syp), enzymes whichcontain two copies of the SH2 domain at its N-terminal extremity (e.g.,the Drosophila protein corkscrew (gene csw) also belongs to thissubgroup), PTPN7 (LC-PTP; Hematopoietic protein-tyrosine phosphatase;HePTP), PTPN8 (70Z-PEP), PTPN9 (MEG2), PTPN12 (PTP-G1; PTP-P19), YeastPTP1, Yeast PTP2 which may be involved in the ubiquitin-mediated proteindegradation pathway, Fission yeast pyp1 and pyp2 which play a role ininhibiting the onset of mitosis, Fission yeast pyp3 which contributes tothe dephosphorylation of cdc2, Yeast CDC14 which may be involved inchromosome segregation, Yersinia virulence plasmid PTPAses (gene yopH),Autographa californica nuclear polyhedrosis virus 19 Kd PTPase, Dualspecificity PTPases, DUSP1 (PTPN10; MAP kinase phosphatase-1; MKP-1);which dephosphorylates MAP kinase on both Thr-183 and Tyr-185, DUSP2(PAC-1), a nuclear enzyme that dephosphorylates MAP kinases ERK1 andERK2 on both Thr and Tyr residues, DUSP3 (VHR), DUSP4 (HVH2), DUSP5(HVH3), DUSP6 (Pyst1; MKP-3), DUSP7 (Pyst2; MKP-X), Yeast MSG5, a PTPasethat dephosphorylates MAP kinase FUS3, Yeast YVH1, Vaccinia virus H1PTPase—a dual specificity phosphatase, Structurally, all known receptorPTPases, are made up of a variable length extracellular domain, followedby a transmembrane region and a C-terminal catalytic cytoplasmic domain.Some of the receptor PTPases contain fibronectin type III (FN-III)repeats, immunoglobulin-like domains, MAM domains or carbonicanhydrase-like domains in their extracellular region. The cytoplasmicregion generally contains two copies of the PTPAse domain. The firstseems to have enzymatic activity, while the second is inactive but seemsto affect substrate specificity of the first. In these domains, thecatalytic cysteine is generally conserved but some other, presumablyimportant, residues are not.

PTPase domains consist of about 300 amino acids. There are two conservedcysteines, the second one has been shown to be absolutely required foractivity. Furthermore, a number of conserved residues in its immediatevicinity have also been shown to be important.

A consensus sequence for tyrosine specific protein phophatases isprovided as follows:

[LIVMF]-H-C-x(2)-G-x(3)-[STC]-[STAGP]-x-[LIVMFY], wherein C is theactive site residue and “X” represents any amino acid.

Additional information related to tyrosine specific protein phosphatasedomains and proteins may be found in reference to the followingpublications Fischer E. H., Charbonneau H., Tonks N. K., Science253:401-406 (1991); Charbonneau H., Tonks N. K., Annu. Rev. Cell Biol.8:463-493 (1992); Trowbridge I. S., J. Biol. Chem. 266:23517-23520(1991); Tonks N. K., Charbonneau H., Trends Biochem. Sci. 14:497-500(1989); and Hunter T., Cell 58:1013-1016 (1989); which are herebyincorporated herein by reference in their entirety.

In preferred embodiments, the following tyrosine specific proteinphosphatase active site domain polypeptide is encompassed by the presentinvention: NGCVLVHCLAGISRSATIAIAYI (SEQ ID NO:144). Polynucleotidesencoding these polypeptides are also provided. The present inventionalso encompasses the use of this tyrosine specific protein phosphataseactive site domain polypeptide as an immunogenic and/or antigenicepitope as described elsewhere herein.

In addition to the human RET31 polynucleotide and polypeptide sequence,the present invention also relates to the isolated mouse ortholog of theRET31 polypeptide.

The polypeptide corresponding to the mouse RET31 gene provided as SEQ IDNO:113 (FIG. 16A-C), encoded by the polynucleotide sequence according toSEQ ID NO:114 (FIG. 16A-C), and/or encoded by the polynucleotidecontained within the deposited clone, mRET31, has significant homologyat the nucleotide and amino acid level to a number of phosphatases,which include, for example, the human RET31 protein of the presentinvention (SEQ ID NO:109); the human DUS8 (DUS8; Genbank AccessionNo:gi|U27193; SEQ ID NO:110); the human DUSP6 protein (DUSP6; GenbankAccession No:gi|AB013382; SEQ ID NO:111); and the human map kinasephosphatase MKP-5 protein (MKP-5; Genbank Accession No:gi|AB026436; SEQID NO:112) as determined by BLASTP. An alignment of the humanphosphatase polypeptide with these proteins is provided in FIGS. 14A-C.

The determined nucleotide sequence of the mRET31 cDNA in FIGS. 16A-C(SEQ ID NO:114) contains an open reading frame encoding a protein ofabout 660 amino acid residues, with a deduced molecular weight of about73 kDa. The amino acid sequence of the predicted mRET31 polypeptide isshown in FIGS. 16A-C (SEQ ID NO:114). The mRET31 protein shown in FIGS.16A-C was determined to share significant identity and similarity toseveral known phosphates, particularly, dual-specificity proteinphosphatases. Specifically, the mRET31 protein shown in FIGS. 16A-C wasdetermined to be about 90% identical and 92% similar to the human RET31protein of the present invention (SEQ ID NO:109); to be about 48.5%identical and 55.7% similar to the human DUS8 (DUS8; Genbank AccessionNo:gi|U27193; SEQ ID NO:110); to be about 37.4% identical and 49.7%similar to the human DUSP6 protein (DUSP6; Genbank AccessionNo:gi|AB013382; SEQ ID NO:111); and to be about 35.2% identical and46.9% similar to the human map kinase phosphatase MKP-5 protein (MKP-5;Genbank Accession No:gi|AB026436; SEQ ID NO:112), as shown in FIG. 12.

The translational start nucleotide position of the mRET31 polynucleotidehas been determined to begin at nucleotide 369 of SEQ ID NO:113 (FIGS.16A-C), and the transational stop nucleotide position has beendetermined to be at nucleotide 2348 of SEQ ID NO:113 (FIGS. 16A-C).

In preferred embodiments, the following N-terminal mRET31 deletionpolypeptides are encompassed by the present invention: M1-S660, A2-S660,H3-S660, E4-S660, M5-S660, I6-S660, G7-S660, T8-S660, Q9-S660, I10-S660,V11-S660, T12-S660, E13-S660, S14-S660, L15-S660, V16-S660, A17-S660,L18-S660, L19-S660, E20-S660, S21-S660, G22-S660, T23-S660, E24-S660,K25-S660, V26-S660, L27-S660, L28-S660, I29-S660, D30-S660, S31-S660,R32-S660, P33-S660, F34-S660, V35-S660, E36-S660, Y37-S660, N38-S660,T39-S660, S40-S660, H41-S660, I42-S660, L43-S660, E44-S660, A45-S660,I46-S660, N47-S660, I48-S660, N49-S660, C50-S660, S51-S660, K52-S660,L53-S660, M54-S660, K55-S660, R56-S660, R57-S660, L58-S660, Q59-S660,Q60-S660, D61-S660, K62-S660, V63-S660, L64-S660, I65-S660, T66-S660,E67-S660, L68-S660, I69-S660, H70-S660, Q71-S660, S72-S660, T73-S660,K74-S660, H75-S660, K76-S660, V77-S660, D78-S660, I79-S660, D80-S660,C81-S660, N82-S660, Q83-S660, R84-S660, V85-S660, V86-S660, V87-S660,Y88-S660, D89-S660, H90-S660, S91-S660, S92-S660, Q93-S660, D94-S660,V95-S660, G96-S660, S97-S660, L98-S660, S99-S660, S100-S660, D101-S660,C102-S660, F103-S660, L104-S660, T105-S660, V106-S660, L107-S660,L108-S660, G109-S660, K110-S660, L111-S660, E112-S660, R113-S660,S114-S660, F115-S660, N116-S660, S117-S660, V118-S660, H119-S660,L120-S660, L121-S660, A122-S660, G123-S660, G124-S660, F125-S660,A126-S660, E127-S660, F128-S660, S129-S660, R130-S660, C131-S660,F132-S660, P133-S660, G134-S660, L135-S660, C136-S660, E137-S660,G138-S660, K139-S660, S140-S660, T141-S660, L142-S660, V143-S660,P144-S660, T145-S660, C146-S660, I147-S660, S148-S660, Q149-S660,P150-S660, C151-S660, L152-S660, P153-S660, V154-S660, A155-S660,N156-S660, I157-S660, G158-S660, P159-S660, T160-S660, R161-S660,I162-S660, L163-S660, P164-S660, N165-S660, L166-S660, Y167-S660,L168-S660, G169-S660, C170-S660, Q171-S660, R172-S660, D173-S660,V174-S660, L175-S660, N176-S660, K177-S660, D178-S660, L179-S660,M180-S660, Q181-S660, Q182-S660, N183-S660, G184-S660, I185-S660,G186-S660, Y187-S660, V188-S660, L189-S660, N190-S660, A191-S660,S192-S660, N193-S660, T194-S660, C195-S660, P196-S660, K197-S660,P198-S660, D199-S660, F200-S660, I201-S660, P202-S660, E203-S660,S204-S660, H205-S660, F206-S660, L207-S660, R208-S660, V209-S660,P210-S660, V211-S660, N212-S660, D213-S660, S214-S660, F215-S660,C216-S660, E217-S660, K218-S660, I219-S660, L220-S660, P221-S660,W222-S660, L223-S660, D224-S660, K225-S660, S226-S660, V227-S660,D228-S660, F229-S660, I230-S660, E231-S660, K232-S660, A233-S660,K634-S660, A235-S660, S236-S660, N237-S660, G238-S660, C239-S660,V240-S660, L241-S660, I242-S660, H243-S660, C244-S660, L245-S660,A246-S660, G247-S660, I248-S660, S249-S660, R250-S660, S251-S660,A252-S660, T253-S660, I254-S660, A255-S660, I256-S660, A257-S660,Y258-S660, I259-S660, M260-S660, K261-S660, R262-S660, M263-S660,D264-S660, M265-S660, S266-S660, L267-S660, D268-S660, E269-S660,A270-S660, Y271-S660, R272-S660, F273-S660, V274-S660, K275-S660,E276-S660, K277-S660, R278-S660, P279-S660, T280-S660, I281-S660,S282-S660, P283-S660, N284-S660, F285-S660, N286-S660, F287-S660,M288-S660, G289-S660, Q290-S660, L291-S660, M292-S660, D293-S660,Y294-S660, E295-S660, K296-S660, T297-S660, I298-S660, N299-S660,N300-S660, Q301-S660, T302-S660, G303-S660, M304-S660, S305-S660,G306-S660, P307-S660, K308-S660, S309-S660, K310-S660, L311-S660,K312-S660, L313-S660, L314-S660, H315-S660, L316-S660, D317-S660,K318-S660, P319-S660, S320-S660, E321-S660, P322-S660, V323-S660,P324-S660, A325-S660, A326-S660, S327-S660, E328-S660, G329-S660,G330-S660, W331-S660, K332-S660, S333-S660, A334-S660, L335-S660,S336-S660, L337-S660, S338-S660, P339-S660, P340-S660, C341-S660,A342-S660, N343-S660, S344-S660, T345-S660, S346-S660, E347-S660,A348-S660, S349-S660, G350-S660, Q351-S660, R352-S660, L353-S660,V354-S660, H355-S660, P356-S660, A357-S660, S358-S660, V359-S660,P360-S660, R361-S660, L362-S660, Q363-S660, P364-S660, S365-S660,L366-S660, L367-S660, E368-S660, D369-S660, S370-S660, P371-S660,L372-S660, V373-S660, Q374-S660, A375-S660, L376-S660, S377-S660,G378-S660, L379-S660, Q380-S660, L381-S660, S382-S660, S383-S660,E384-S660, K385-S660, L386-S660, E387-S660, D388-S660, S389-S660,T390-S660, K391-S660, L392-S660, K393-S660, R394-S660, S395-S660,F396-S660, S397-S660, L398-S660, D399-S660, I400-S660, K401-S660,S402-S660, V403-S660, S404-S660, Y405-S660, S406-S660, A407-S660,S408-S660, M409-S660, A410-S660, A411-S660, S412-S660, L413-S660,H414-S660, G415-S660, F416-S660, S417-S660, S418-S660, E419-S660,E420-S660, A421-S660, L422-S660, D423-S660, Y424-S660, C425-S660,K426-S660, P427-S660, S428-S660, A429-S660, T430-S660, L431-S660,D432-S660, G433-S660, T434-S660, N435-S660, K436-S660, L437-S660,C438-S660, Q439-S660, F440-S660, S441-S660, P442-S660, V443-S660,Q444-S660, E445-S660, V446-S660, S447-S660, E448-S660, Q449-S660,S450-S660, P451-S660, E452-S660, T453-S660, S454-S660, P455-S660,D456-S660, K457-S660, E458-S660, E459-S660, A460-S660, H461-S660,I462-S660, P463-S660, K464-S660, Q465-S660, P466-S660, Q467-S660,P468-S660, P469-S660, R470-S660, P471-S660, S472-S660, E473-S660,S474-S660, Q475-S660, V476-S660, T477-S660, R478-S660, L479-S660,H480-S660, S481-S660, V482-S660, R483-S660, T484-S660, G485-S660,S486-S660, S487-S660, G488-S660, S489-S660, T490-S660, Q491-S660,R492-S660, P493-S660, F494-S660, F495-S660, S496-S660, P497-S660,L498-S660, H499-S660, R500-S660, S501-S660, G502-S660, S503-S660,V504-S660, E505-S660, D506-S660, N507-S660, Y508-S660, H509-S660,T510-S660, N511-S660, F512-S660, L513-S660, F514-S660, G515-S660,L516-S660, S517-S660, T518-S660, S519-S660, Q520-S660, Q521-S660,H522-S660, L523-S660, T524-S660, K525-S660, S526-S660, A527-S660,G528-S660, L529-S660, G530-S660, L531-S660, K532-S660, G533-S660,W534-S660, H535-S660, S536-S660, D537-S660, I538-S660, L539-S660,A540-S660, P541-S660, Q542-S660, S543-S660, S544-S660, A545-S660,P546-S660, S547-S660, L548-S660, T549-S660, S550-S660, S551-S660,W552-S660, Y553-S660, F554-S660, A555-S660, T556-S660, E557-S660,P558-S660, S559-S660, H560-S660, L561-S660, Y562-S660, S563-S660,A564-S660, S565-S660, A566-S660, I567-S660, Y568-S660, G569-S660,G570-S660, N571-S660, S572-S660, S573-S660, Y574-S660, S575-S660,A576-S660, Y577-S660, S578-S660, C579-S660, G580-S660, Q581-S660,L582-S660, P583-S660, T584-S660, C585-S660, S586-S660, D587-S660,Q588-S660, I589-S660, Y590-S660, S591-S660, V592-S660, R593-S660,R594-S660, R595-S660, Q596-S660, K597-S660, P598-S660, T599-S660,D600-S660, R601-S660, A602-S660, D603-S660, S604-S660, R605-S660,R606-S660, S607-S660, W608-S660, H609-S660, E610-S660, E611-S660,S612-S660, P613-S660, F614-S660, E615-S660, K616-S660, Q617-S660,F618-S660, K619-S660, R620-S660, R621-S660, S622-S660, C623-S660,Q624-S660, M625-S660, E626-S660, F627-S660, G628-S660, E629-S660,S630-S660, I631-S660, M632-S660, S633-S660, E634-S660, N635-S660,R636-S660, S637-S660, R638-S660, E639-S660, E640-S660, L641-S660,G642-S660, K643-S660, V644-S660, G645-S660, S646-S660, Q647-S660,S648-S660, S649-S660, F650-S660, S651-S660, G652-S660, S653-S660, and/orM654-S660 of SEQ ID NO:114. Polynucleotide sequences encoding thesepolypeptides are also provided. The present invention also encompassesthe use of these N-terminal mRET31 deletion polypeptides as immunogenicand/or antigenic epitopes as described elsewhere herein.

In preferred embodiments, the following C-terminal mRET31 deletionpolypeptides are encompassed by the present invention: M1-S660, M1-V659,M1-E658, M1-I657, M1-I656, M1-E655, M1-M654, M1-S653, M1-G652, M1-S651,M1-F650, M1-S649, M1-S648, M1-Q647, M1-S646, M1-G645, M1-V644, M1-K643,M1-G642, M1-L641, M1-E640, M1-E639, M1-R638, M1-S637, M1-R636, M1-N635,M1-E634, M1-S633, M1-M632, M1-I631, M1-S630, M1-E629, M1-G628, M1-F627,M1-E626, M1-M625, M1-Q624, M1-C623, M1-S622, M1-R621, M1-R620, M1-K619,M1-F618, M1-Q617, M1-K616, M1-E615, M1-F614, M1-P613, M1-S612, M1-E611,M1-E610, M1-H609, M1-W608, M1-S607, M1-R606, M1-R605, M1-S604, M1-D603,M1-A602, M1-R601, M1-D600, M1-T599, M1-P598, M1-K597, M1-Q596, M1-R595,M1-R594, M1-R593, M1-V592, M1-S591, M1-Y590, M1-I589, M1-Q588, M1-D587,M1-S586, M1-C585, M1-T584, M1-P583, M1-L582, M1-Q581, M1-G580, M1-C579,M1-S578, M1-Y577, M1-A576, M1-S575, M1-Y574, M1-S573, M1-S572, M1-N571,M1-G570, M1-G569, M1-Y568, M1-I567, M1-A566, M1-S565, M1-A564, M1-S563,M1-Y562, M1-L561, M1-H560, M1-S559, M1-P558, M1-E557, M1-T556, M1-A555,M1-F554, M1-Y553, M1-W552, M1-S551, M1-S550, M1-T549, M1-L548, M1-S547,M1-P546, M1-A545, M1-S544, M1-S543, M1-Q542, M1-P541, M1-A540, M1-L539,M1-I538, M1-D537, M1-S536, M1-H535, M1-W534, M1-G533, M1-K532, M1-L531,M1-G530, M1-L529, M1-G528, M1-A527, M1-S526, M1-K525, M1-T524, M1-L523,M1-H522, M1-Q521, M1-Q520, M1-S519, M1-T518, M1-S517, M1-L516, M1-G515,M1-F514, M1-L513, M1-F512, M1-N511, M1-T510, M1-H509, M1-Y508, M1-N507,M1-D506, M1-E505, M1-V504, M1-S503, M1-G502, M1-S501, M1-R500, M1-H499,M1-L498, M1-P497, M1-S496, M1-F495, M1-F494, M1-P493, M1-R492, M1-Q491,M1-T490, M1-S489, M1-G488, M1-S487, M1-S486, M1-G485, M1-T484, M1-R483,M1-V482, M1-S481, M1-H480, M1-L479, M1-R478, M1-T477, M1-V476, M1-Q475,M1-S474, M1-E473, M1-S472, M1-P471, M1-R470, M1-P469, M1-P468, M1-Q467,M1-P466, M1-Q465, M1-K464, M1-P463, M1-I462, M1-H461, M1-A460, M1-E459,M1-E458, M1-K457, M1-D456, M1-P455, M1-S454, M1-T453, M1-E452, M1-P451,M1-S450, M1-Q449, M1-E448, M1-S447, M1-V446, M1-E445, M1-Q444, M1-V443,M1-P442, M1-S441, M1-F440, M1-Q439, M1-C438, M1-L437, M1-K436, M1-N435,M1-T434, M1-G433, M1-D432, M1-L431, M1-T430, M1-A429, M1-S428, M1-P427,M1-K426, M1-C425, M1-Y424, M1-D423, M1-L422, M1-A421, M1-E420, M1-E419,M1-S418, M1-S417, M1-F416, M1-G415, M1-H414, M1-L413, M1-S412, M1-A411,M1-A410, M1-M409, M1-S408, M1-A407, M1-S406, M1-Y405, M1-S404, M1-V403,M1-S402, M1-K401, M1-I400, M1-D399, M1-L398, M1-S397, M1-F396, M1-S395,M1-R394, M1-K393, M1-L392, M1-K391, M1-T390, M1-S389, M1-D388, M1-E387,M1-L386, M1-K385, M1-E384, M1-S383, M1-S382, M1-L381, M1-Q380, M1-L379,M1-G378, M1-S377, M1-L376, M1-A375, M1-Q374, M1-V373, M1-L372, M1-P371,M1-S370, M1-D369, M1-E368, M1-L367, M1-L366, M1-S365, M1-P364, M1-Q363,M1-L362, M1-R361, M1-P360, M1-V359, M1-S358, M1-A357, M1-P356, M1-H355,M1-V354, M1-L353, M1-R352, M1-Q351, M1-G350, M1-S349, M1-A348, M1-E347,M1-S346, M1-T345, M1-S344, M1-N343, M1-A342, M1-C341, M1-P340, M1-P339,M1-S338, M1-L337, M1-S336, M1-L335, M1-A334, M1-S333, M1-K332, M1-W331,M1-G330, M1-G329, M1-E328, M1-S327, M1-A326, M1-A325, M1-P324, M1-V323,M1-P322, M1-E321, M1-S320, M1-P319, M1-K318, M1-D317, M1-L316, M1-H315,M1-L314, M1-L313, M1-K312, M1-L311, M1-K310, M1-S309, M1-K308, M1-P307,M1-G306, M1-S305, M1-M304, M1-G303, M1-T302, M1-Q301, M1-N300, M1-N299,M1-I298, M1-T297, M1-K296, M1-E295, M1-Y294, M1-D293, M1-M292, M1-L291,M1-Q290, M1-G289, M1-M288, M1-F287, M1-N286, M1-F285, M1-N284, M1-P283,M1-S282, M1-I281, M1-T280, M1-P279, M1-R278, M1-K277, M1-E276, M1-K275,M1-V274, M1-F273, M1-R272, M1-Y271, M1-A270, M1-E269, M1-D268, M1-L267,M1-S266, M1-M265, M1-D264, M1-M263, M1-R262, M1-K261, M1-M260, M1-I259,M1-Y258, M1-A257, M1-I256, M1-A255, M1-I254, M1-T253, M1-A252, M1-S251,M1-R250, M1-S249, M1-I248, M1-G247, M1-A246, M1-L245, M1-C244, M1-H243,M1-I242, M1-L241, M1-V240, M1-C239, M1-G238, M1-N237, M1-S236, M1-A235,M1-K234, M1-A233, M1-K232, M1-E231, M1-I230, M1-F229, M1-D228, M1-V227,M1-S226, M1-K225, M1-D224, M1-L223, M1-W222, M1-P221, M1-L220, M1-I219,M1-K218, M1-E217, M1-C216, M1-F215, M1-S214, M1-D213, M1-N212, M1-V211,M1-P210, M1-V209, M1-R208, M1-L207, M1-F206, M1-H205, M1-S204, M1-E203,M1-P202, M1-I201, M1-F200, M1-D199, M1-P198, M1-K197, M1-P196, M1-C195,M1-T194, M1-N193, M1-S192, M1-A191, M1-N190, M1-L189, M1-V188, M1-Y187,M1-G186, M1-I185, M1-G184, M1-N183, M1-Q182, M1-Q181, M1-M180, M1-L179,M1-D178, M1-K177, M1-N176, M1-L175, M1-V174, M1-D173, M1-R172, M1-Q171,M1-C170, M1-G169, M1-L168, M1-Y167, M1-L166, M1-N165, M1-P164, M1-L163,M1-I162, M1-R161, M1-T160, M1-P159, M1-G158, M1-I157, M1-N156, M1-A155,M1-V154, M1-P153, M1-L152, M1-C151, M1-P150, M1-Q149, M1-S148, M1-I147,M1-C146, M1-T145, M1-P144, M1-V143, M1-L142, M1-T141, M1-S140, M1-K139,M1-G138, M1-E137, M1-C136, M1-L135, M1-G134, M1-P133, M1-F132, M1-C131,M1-R130, M1-S129, M1-F128, M1-E127, M1-A126, M1-F125, M1-G124, M1-G123,M1-A122, M1-L121, M1-L120, M1-H119, M1-V118, M1-S117, M1-N116, M1-F115,M1-S114, M1-R113, M1-E112, M1-L111, M1-K110, M1-G109, M1-L108, M1-L107,M1-V106, M1-T105, M1-L104, M1-F103, M1-C102, M1-D101, M1-S100, M1-S99,M1-L98, M1-S97, M1-G96, M1-V95, M1-D94, M1-Q93, M1-S92, M1-S91, M1-H90,M1-D89, M1-Y88, M1-V87, M1-V86, M1-V85, M1-R84, M1-Q83, M1-N82, M1-C81,M1-D80, M1-I79, M1-D78, M1-V77, M1-K76, M1-H75, M1-K74, M1-T73, M1-S72,M1-Q71, M1-H70, M1-I69, M1-L68, M1-E67, M1-T66, M1-I65, M1-L64, M1-V63,M1-K62, M1-D61, M1-Q60, M1-Q59, M1-L58, M1-R57, M1-R56, M1-K55, M1-M54,M1-L53, M1-K52, M1-S51, M1-C50, M1-N49, M1-I48, M1-N47, M1-I46, M1-A45,M1-E44, M1-L43, M1-I42, M1-H41, M1-S40, M1-T39, M1-N38, M1-Y37, M1-E36,M1-V35, M1-F34, M1-P33, M1-R32, M1-S31, M1-D30, M1-I29, M1-L28, M1-L27,M1-V26, M1-K25, M1-E24, M1-T23, M1-G22, M1-S21, M1-E20, M1-L19, M1-L18,M1-A17, M1-V16, M1-L15, M1-S14, M1-E13, M1-T12, M1-V11, M1-I10, M1-Q9,M1-T8, and/or M1-G7 of SEQ ID NO:114. Polynucleotide sequences encodingthese polypeptides are also provided. The present invention alsoencompasses the use of these C-terminal mRET31 deletion polypeptides asimmunogenic and/or antigenic epitopes as described elsewhere herein.

In confirmation of the mouse RET31 representing a novel mousephosphatase polypeptide, the mRET31 polypeptide has been shown tocomprise a dual specificity phosphatase catalytic domain as identifiedby the BLAST2 algorithm using the DSPc PFAM HMM (PF00782) as a querysequence.

In preferred embodiments, the following mouse RET31 DSPc domainpolypeptide is encompassed by the present invention:GPTRILPNLYLGCQRDVLNKDLMQQNGIGYVLNASNTCPKPDFIPESHFLRVPVNDSFCEKILPWLDKSVDFIEKAKASNGCVLIHCLAGISRSATIAIAYIMKRMDMSLDEAYRFVKEKRPTISPNFNFMGQLMDYEKT (SEQ ID NO:135). Polynucleotides encoding thispolypeptide are also provided. The present invention also encompassesthe use of this mouse RET31 DSPc domain polypeptide as an immunogenicand/or antigenic epitope as described elsewhere herein.

The present invention encompasses the use of RET31 inhibitors and/oractivators of RET31 activity for the treatment, detectoin,amelioaration, or prevention of phosphatase associated disorders,including but not limited to metabolic diseases such as diabetes, inaddition to neural and/or cardiovascular diseases and disorders. Thepresent invention also encompasses the use of RET31 inhibitors and/oractivators of RET31 activity as immunosuppressive agents,anti-inflammatory agents, and/or anti-tumor agents

The present invention encompasses the use of RET31 phosphataseinhibitors, including, antagonists such as antisense nucleic acids, inaddition to other antagonists, as described herein, in a therapeuticregimen to diagnose, prognose, treat, ameliorate, and/or preventdiseases where a kinase activity is insufficient. One, non-limitingexample of a disease which may occur due to insufficient kinase activityare certain types of diabetes, where one or more kinases involved in theinsulin receptor signal pathway may have insufficient activity orinsufficient expression, for example.

Moreover, the present invention encompasses the use of RET31 phosphataseactivators, and/or the use of the RET31 phosphatase gene or protein in agene therapy regimen, as described herein, for the diagnoses, prognoses,treatment, amelioration, and/or prevention of diseases and/or disorderswhere a kinase activity is overly high, such as a cancer where a kinaseoncogene product has excessive activity or excessive expression.

The present invention also encompasses the use of catalytically inactivevariants of RET31 proteins, including fragments thereof, such as aprotein therapeutic, or the use of the encoding polynucleotide sequenceor as gene therapy, for example, in the diagnoses, prognosis, treatment,amelioration, and/or prevention of diseases or disorders wherephosphatase activity is overly high.

The present invention encompasses the use of antibodies directed againstthe RET31 polypeptides, including fragment and/or variants thereof, ofthe present invention in diagnostics, as a biomarkers, and/or as atherapeutic agents.

The present invention encompasses the use of an inactive, non-catalytic,mutant of the RET31 phosphatase as a substrate trapping mutant to bindcellular phosphoproteins or a library of phosphopeptides to identifysubstrates of the RET31 polypeptides.

The present invention encompasses the use of the RET31 polypeptides, toidentify inhibitors or activators of the RET31 phosphatase activityusing either in vitro or ‘virtual’ (in silico) screening methods.

One embodiment of the invention relates to a method for identifying acompound as an activator or inhibitor of the RET31 phosphatasecomprising the steps of: i.) contacting a RET31 phosphatase inhibitor oractivator labeled with an analytically detectable reagent with the RET31phosphatase under conditions sufficient to form a complex with theinhibitor or activator; ii.) contacting said complex with a samplecontaining a compound to be identified; iii) and identifying thecompound as an inhibitor or activator by detecting the ability of thetest compound to alter the amount of labeled known RET31 phosphataseinhibitor or activator in the complex.

Another embodiment of the invention relates to a method for identifyinga compound as an activator or inhibitor of a RET31 phosphatasecomprising the steps of: i.) contacting the RET31 phosphatase with acompound to be identified; and ii.) and measuring the ability of theRET31 phosphatase to remove phosphate from a substrate.

The present invention also encomposses a method for identifying a ligandfor the RET31 phosphatase comprising the steps of: i.) contacting theRET31 phosphatase with a series of compounds under conditions to permitbinding; and ii.) detecting the presence of any ligand-bound protein.

Preferably, the above referenced methods comprise the RET31 phosphatasein a form selected from the group consisting of whole cells, cytosoliccell fractions, membrane cell fractions, purified or partially purifiedforms. The invention also relates to recombinantly expressed RET31phosphatase in a purified, substantially purified, or unpurified state.The invention further relates to RET31 phosphatase fused or conjugatedto a protein, peptide, or other molecule or compound known in the art,or referenced herein.

The present invention also encompasses pharmaceutical composition of theRET31 phosphatase polypeptide comprising a compound identified by abovereferenced methods and a pharmaceutically acceptable carrier.

In preferred embodiments, the present invention encompasses apolynucleotide lacking the initiating start codon, in addition to, theresulting encoded polypeptide of RET31. Specifically, the presentinvention encompasses the polynucleotide corresponding to nucleotides541 thru 2532 of SEQ ID NO:108, and the polypeptide corresponding toamino acids 2 thru 665 of SEQ ID NO:109. Also encompassed arerecombinant vectors comprising said encoding sequence, and host cellscomprising said vector.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO: 108 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides consisting of a nucleotide sequence described bythe general formula of a−b, where a is any integer between 1 to 5436 ofSEQ ID NO:108, b is an integer between 15 to 5450, where both a and bcorrespond to the positions of nucleotide residues shown in SEQ IDNO:108, and where b is greater than or equal to a +14.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO: 113 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides consisting of a nucleotide sequence described bythe general formula of a−b, where a is any integer between 1 to 2742 ofSEQ ID NO:113, b is an integer between 15 to 2756, where both a and bcorrespond to the positions of nucleotide residues shown in SEQ IDNO:113, and where b is greater than or equal to a+14.

TABLE I ATCC Deposit Total 5′ NT 3′ Total No. Z NT NT of Start NT AA AAGene CDNA and SEQ Seq of Codon of Seq ID of No. CloneID Date Vector ID.No. X Clone of ORF ORF No. Y ORF 1. BMY_HP Xxxxxx 149 4393 628 2448 150607 P1_FL Xx/xx/xx 1. BMY_HP Xxxxxx 1 144 1 144 2 48 P1 - Xx/xx/xxFragment A 1. BMY_HP Xxxxxx 3 33 1 33 4 11 P1 - Xx/xx/xx Fragment B 2.BMY_HP Xxxxxx 151 878 89 538 152 150 P2_FL Xx/xx/xx 2. BMY_HP Xxxxxx 5746 2 745 6 248 P2_partial Xx/xx/xx 3. BMY_HP Xxxxxx 7 511 1 510 8 170P3 Xx/xx/xx 4. BMY_HP Xxxxxx 9 1710 1 1710 10 570 P4 Xx/xx/xx 5. BMY_HPPTA- pSport 41 5111 470 2464 42 665 P5 (7IC-5- 2966 E2) Jan. 24, 2001 6.RET31 PTA- PTA 108 5450 538 2532 109 665 (also 3434 dv referred to Jun.07, 2001 as as 1hrTNF031, and/or Clone 31

Table I summarizes the information corresponding to each “Gene No.”described above. The nucleotide sequence identified as “NT SEQ ID NO:X”was assembled from partially homologous (“overlapping”) sequencesobtained from the “cDNA clone ID” identified in Table I and, in somecases, from additional related DNA clones. The overlapping sequenceswere assembled into a single contiguous sequence of high redundancy(usually several overlapping sequences at each nucleotide position),resulting in a final sequence identified as SEQ ID NO:X.

The cDNA Clone ID was deposited on the date and given the correspondingdeposit number listed in “ATCC Deposit No:Z and Date.” “Vector” refersto the type of vector contained in the cDNA Clone ID.

“Total NT Seq. Of Clone” refers to the total number of nucleotides inthe clone-contig identified by “Gene No.” The deposited clone maycontain all or most of the sequence of SEQ ID NO:X. The nucleotideposition of SEQ ID NO:X of the putative start codon (methionine) isidentified as “5′ NT of Start Codon of ORF.”

The translated amino acid sequence, beginning with the methionine, isidentified as “AA SEQ ID NO:Y,” although other reading frames can alsobe easily translated using known molecular biology techniques. Thepolypeptides produced by these alternative open reading frames arespecifically contemplated by the present invention.

The total number of amino acids within the open reading frame of SEQ IDNO:Y is identified as “Total AA of ORF”.

SEQ ID NO:X (where X may be any of the polynucleotide sequencesdisclosed in the sequence listing) and the translated SEQ ID NO:Y (whereY may be any of the polypeptide sequences disclosed in the sequencelisting) are sufficiently accurate and otherwise suitable for a varietyof uses well known in the art and described further herein. Forinstance, SEQ ID NO:X is useful for designing nucleic acid hybridizationprobes that will detect nucleic acid sequences contained in SEQ ID NO:Xor the cDNA contained in the deposited clone. These probes will alsohybridize to nucleic acid molecules in biological samples, therebyenabling a variety of forensic and diagnostic methods of the invention.Similarly, polypeptides identified from SEQ ID NO:Y may be used, forexample, to generate antibodies which bind specifically to proteinscontaining the polypeptides and the proteins encoded by the cDNA clonesidentified in Table I.

Nevertheless, DNA sequences generated by sequencing reactions cancontain sequencing errors. The errors exist as misidentifiednucleotides, or as insertions or deletions of nucleotides in thegenerated DNA sequence. The erroneously inserted or deleted nucleotidesmay cause frame shifts in the reading frames of the predicted amino acidsequence. In these cases, the predicted amino acid sequence divergesfrom the actual amino acid sequence, even though the generated DNAsequence may be greater than 99.9% identical to the actual DNA sequence(for example, one base insertion or deletion in an open reading frame ofover 1000 bases).

Accordingly, for those applications requiring precision in thenucleotide sequence or the amino acid sequence, the present inventionprovides not only the generated nucleotide sequence identified as SEQ IDNO:X and the predicted translated amino acid sequence identified as SEQID NO:Y, but also a sample of plasmid DNA containing a cDNA of theinvention deposited with the ATCC, as set forth in Table I. Thenucleotide sequence of each deposited clone can readily be determined bysequencing the deposited clone in accordance with known methods. Thepredicted amino acid sequence can then be verified from such deposits.Moreover, the amino acid sequence of the protein encoded by a particularclone can also be directly determined by peptide sequencing or byexpressing the protein in a suitable host cell containing the depositedcDNA, collecting the protein, and determining its sequence.

The present invention also relates to the genes corresponding to SEQ IDNO:X, SEQ ID NO:Y, or the deposited clone. The corresponding gene can beisolated in accordance with known methods using the sequence informationdisclosed herein. Such methods include preparing probes or primers fromthe disclosed sequence and identifying or amplifying the correspondinggene from appropriate sources of genomic material.

Also provided in the present invention are species homologs, allelicvariants, and/or orthologs. The skilled artisan could, using procedureswell-known in the art, obtain the polynucleotide sequence correspondingto full-length genes (including, but not limited to the full-lengthcoding region), allelic variants, splice variants, orthologs, and/orspecies homologues of genes corresponding to SEQ ID NO:X, SEQ ID NO:Y,or a deposited clone, relying on the sequence from the sequencesdisclosed herein or the clones deposited with the ATCC. For example,allelic variants and/or species homologues may be isolated andidentified by making suitable probes or primers which correspond to the5′, 3′, or internal regions of the sequences provided herein andscreening a suitable nucleic acid source for allelic variants and/or thedesired homologue.

The polypeptides of the invention can be prepared in any suitablemanner. Such polypeptides include isolated naturally occurringpolypeptides, recombinantly produced polypeptides, syntheticallyproduced polypeptides, or polypeptides produced by a combination ofthese methods. Means for preparing such polypeptides are well understoodin the art.

The polypeptides may be in the form of the protein, or may be a part ofa larger protein, such as a fusion protein (see below). It is oftenadvantageous to include an additional amino acid sequence which containssecretory or leader sequences, pro-sequences, sequences which aid inpurification, such as multiple histidine residues, or an additionalsequence for stability during recombinant production.

The polypeptides of the present invention are preferably provided in anisolated form, and preferably are substantially purified. Arecombinantly produced version of a polypeptide, can be substantiallypurified using techniques described herein or otherwise known in theart, such as, for example, by the one-step method described in Smith andJohnson, Gene 67:31-40 (1988). Polypeptides of the invention also can bepurified from natural, synthetic or recombinant sources using protocolsdescribed herein or otherwise known in the art, such as, for example,antibodies of the invention raised against the full-length form of theprotein.

The present invention provides a polynucleotide comprising, oralternatively consisting of, the sequence identified as SEQ ID NO:X,and/or a cDNA provided in ATCC Deposit No. Z:. The present inventionalso provides a polypeptide comprising, or alternatively consisting of,the sequence identified as SEQ ID NO:Y, and/or a polypeptide encoded bythe cDNA provided in ATCC Deposit NO:Z. The present invention alsoprovides polynucleotides encoding a polypeptide comprising, oralternatively consisting of the polypeptide sequence of SEQ ID NO:Y,and/or a polypeptide sequence encoded by the cDNA contained in ATCCDeposit No:Z.

Preferably, the present invention is directed to a polynucleotidecomprising, or alternatively consisting of, the sequence identified asSEQ ID NO:X, and/or a cDNA provided in ATCC Deposit No.: that is lessthan, or equal to, a polynucleotide sequence that is 5 mega basepairs, 1mega basepairs, 0.5 mega basepairs, 0.1 mega basepairs, 50,000basepairs, 20,000 basepairs, or 10,000 basepairs in length.

The present invention encompasses polynucleotides with sequencescomplementary to those of the polynucleotides of the present inventiondisclosed herein. Such sequences may be complementary to the sequencedisclosed as SEQ ID NO:X, the sequence contained in a deposit, and/orthe nucleic acid sequence encoding the sequence disclosed as SEQ IDNO:Y.

The present invention also encompasses polynucleotides capable ofhybridizing, preferably under reduced stringency conditions, morepreferably under stringent conditions, and most preferably under highlystringent conditions, to polynucleotides described herein. Examples ofstringency conditions are shown in Table II below: highly stringentconditions are those that are at least as stringent as, for example,conditions A-F; stringent conditions are at least as stringent as, forexample, conditions G-L; and reduced stringency conditions are at leastas stringent as, for example, conditions M-R.

TABLE II Hybridization Wash Stringency Polynucleotide Hybrid TemperatureTemperature Condition Hybrid± Length (bp)‡ and Buffer† and Buffer† ADNA:DNA > or equal to 50 65° C.; 1xSSC - 65° C.; 0.3xSSC or- 42° C.;1xSSC, 50% formamide B DNA:DNA <50 Tb*; 1xSSC Tb*; 1xSSC C DNA:RNA > orequal to 50 67° C.; 1xSSC - 67° C.; 0.3xSSC or- 45° C.; 1xSSC, 50%formamide D DNA:RNA <50 Td*; 1xSSC Td*; 1xSSC E RNA:RNA > or equal to70° C.; 1xSSC - 70° C.; 0.3xSSC 50 or- 50° C.; 1xSSC, 50% formamide FRNA:RNA <50 Tf*; 1xSSC Tf*; 1xSSC G DNA:DNA > or equal to 65° C.;4xSSC - 65° C.; 1xSSC 50 or- 45° C.; 4xSSC, 50% formamide H DNA:DNA <50Th*; 4xSSC Th*; 4xSSC I DNA:RNA > or equal to 67° C.; 4xSSC - 67° C.;1xSSC 50 or- 45° C.; 4xSSC, 50% formamide J DNA:RNA <50 Tj*; 4xSSC Tj*;4xSSC K RNA:RNA > or equal to 70° C.; 4xSSC - 67° C.; 1xSSC 50 or- 40°C.; 6xSSC, 50% formamide L RNA:RNA <50 Tl*; 2xSSC Tl*; 2xSSC M DNA:DNA >or equal to 50° C.; 4xSSC - 50° C.; 2xSSC 50 or- 40° C. 6xSSC, 50%formamide N DNA:DNA <50 Tn*; 6xSSC Tn*; 6xSSC O DNA:RNA > or equal to55° C.; 4xSSC - 55° C.; 2xSSC 50 or- 42° C.; 6xSSC, 50% formamide PDNA:RNA <50 Tp*; 6xSSC Tp*; 6xSSC Q RNA:RNA > or equal to 60° C.;4xSSC - 60° C.; 2xSSC 50 or- 45° C.; 6xSSC, 50% formamide R RNA:RNA <50Tr*; 4xSSC Tr*;4xSSC ‡The “hybrid length” is the anticipated length forthe hybridized region(s) of the hybridizing polynucleotides. Whenhybridizing a polynucleotide of unknown sequence, the hybrid is assumedto be that of the hybridizing polynucleotide of the present invention.Whenpolynucleotides of known sequence are hybridized, the hybrid lengthcan be determined by aligning the sequences of the polynucleotides andidentifying the region or regions of optimal sequence complementarity.Methods of aligning two or more polynucleotide sequencesand/ordetermining the percent identity between two polynucleotidesequences are well known in the art (e.g., MegAlign program of theDNA*Star suite of programs, etc.) †SSPE (1xSSPE is 0.15M NaCl, 10 mMNaH2PO4, and 1.25 mM EDTA, pH 7.4) can be substituted for SSC (1xSSC is0.15M NaCl and 15 mM sodium citrate) in the hybridization and washbuffers; washes are performed for 15 minutes after hybridizationiscomplete. The hybridizations and washes may additionally include 5XDenhardt's reagent, .5-1.0% SDS, 100 ug/ml denatured, fragmented salmonsperm DNA, 0.5% sodium pyrophosphate, and up to 50% formamide. *Tb-Tr:The hybridization temperature for hybrids anticipated to be less than 50base pairs in length should be 5-10° C. less than the meltingtemperature Tm of the hybrids there Tm is determined according to thefollowing equations. For hybrids less than 18 basepairs in length, Tm(°C.) = 2(# of A + T bases) + 4(# of G + C bases). For hybrids between 18and 49 base pairs in length, Tm(° C.) = 81.5 + 16.6(log₁₀[Na+]) + 0.41(%G + C) − (600/N), where N isthe number of bases in the hybrid, and [Na+]is the concentration of sodium ions in the hybridization buffer ([Na+]for 1xSSC = .165 M). ±The present invention encompasses the substitutionof any one, or more DNA or RNA hybrid partners with either a PNA, or amodified polynucleotide. Such modifiedpolynucleotides are known in theart and are more particularly described elsewhere herein.

Additional examples of stringency conditions for polynucleotidehybridization are provided, for example, in Sambrook, J., E. F. Fritsch,and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and11, and Current Protocols in Molecular Biology, 1995, F. M., Ausubel etal., eds, John Wiley and Sons, Inc., sections 2.10 and 6.3-6.4, whichare hereby incorporated by reference herein.

Preferably, such hybridizing polynucleotides have at least 70% sequenceidentity (more preferably, at least 80% identity; and most preferably atleast 90% or 95% identity) with the polynucleotide of the presentinvention to which they hybridize, where sequence identity is determinedby comparing the sequences of the hybridizing polynucleotides whenaligned so as to maximize overlap and identity while minimizing sequencegaps. The determination of identity is well known in the art, anddiscussed more specifically elsewhere herein.

The invention encompasses the application of PCR methodology to thepolynucleotide sequences of the present invention, the clone depositedwith the ATCC, and/or the cDNA encoding the polypeptides of the presentinvention. PCR techniques for the amplification of nucleic acids aredescribed in U.S. Pat. No. 4,683,195 and Saiki et al., Science,239:487-491 (1988). PCR, for example, may include the following steps,of denaturation of template nucleic acid (if double-stranded), annealingof primer to target, and polymerization. The nucleic acid probed or usedas a template in the amplification reaction may be genomic DNA, cDNA,RNA, or a PNA. PCR may be used to amplify specific sequences fromgenomic DNA, specific RNA sequence, and/or cDNA transcribed from mRNA.References for the general use of PCR techniques, including specificmethod parameters, include Mullis et al., Cold Spring Harbor Symp.Quant. Biol., 51:263, (1987), Ehrlich (ed), PCR Technology, StocktonPress, NY, 1989; Ehrlich et al., Science, 252:1643-1650, (1991); and“PCR Protocols, A Guide to Methods and Applications”, Eds., Innis etal., Academic Press, New York, (1990).

Signal Sequences

The present invention also encompasses mature forms of the polypeptidecomprising, or alternatively consisting of, the polypeptide sequence ofSEQ ID NO:Y, the polypeptide encoded by the polynucleotide described asSEQ ID NO:X, and/or the polypeptide sequence encoded by a cDNA in thedeposited clone. The present invention also encompasses polynucleotidesencoding mature forms of the present invention, such as, for example thepolynucleotide sequence of SEQ ID NO:X, and/or the polynucleotidesequence provided in a cDNA of the deposited clone.

According to the signal hypothesis, proteins secreted by eukaryoticcells have a signal or secretary leader sequence which is cleaved fromthe mature protein once export of the growing protein chain across therough endoplasmic reticulum has been initiated. Most eukaryotic cellscleave secreted proteins with the same specificity. However, in somecases, cleavage of a secreted protein is not entirely uniform, whichresults in two or more mature species of the protein. Further, it haslong been known that cleavage specificity of a secreted protein isultimately determined by the primary structure of the complete protein,that is, it is inherent in the amino acid sequence of the polypeptide.

Methods for predicting whether a protein has a signal sequence, as wellas the cleavage point for that sequence, are available. For instance,the method of McGeoch, Virus Res. 3:271-286 (1985), uses the informationfrom a short N-terminal charged region and a subsequent uncharged regionof the complete (uncleaved) protein. The method of von Heinje, NucleicAcids Res. 14:4683-4690 (1986) uses the information from the residuessurrounding the cleavage site, typically residues −13 to +2, where +1indicates the amino terminus of the secreted protein. The accuracy ofpredicting the cleavage points of known mammalian secretory proteins foreach of these methods is in the range of 75-80%. (von Heinje, supra.)However, the two methods do not always produce the same predictedcleavage point(s) for a given protein.

The established method for identifying the location of signal sequences,in addition, to their cleavage sites has been the SignalP program (v1.1)developed by Henrik Nielsen et al., Protein Engineering 10:1-6 (1997).The program relies upon the algorithm developed by von Heinje, thoughprovides additional parameters to increase the prediction accuracy.

More recently, a hidden Markov model has been developed (H. Neilson, etal., Ismb 1998; 6:122-30), which has been incorporated into the morerecent SignalP (v2.0). This new method increases the ability to identifythe cleavage site by discriminating between signal peptides anduncleaved signal anchors. The present invention encompasses theapplication of the method disclosed therein to the prediction of thesignal peptide location, including the cleavage site, to any of thepolypeptide sequences of the present invention.

As one of ordinary skill would appreciate, however, cleavage sitessometimes vary from organism to organism and cannot be predicted withabsolute certainty. Accordingly, the polypeptide of the presentinvention may contain a signal sequence. Polypeptides of the inventionwhich comprise a signal sequence have an N-terminus beginning within 5residues (i.e., + or −5 residues, or preferably at the −5, −4, −3, −2,−1, +1, +2, +3, +4, or +5 residue) of the signal sequence from asecreted protein is not entirely uniform, resulting in more than onesecreted species. These polypeptides, and the polynucleotides encodingsuch polypeptides, are contemplated by the present invention.

Moreover, the signal sequence identified by the above analysis may notnecessarily predict the naturally occurring signal sequence. Forexample, the naturally occurring signal sequence may be further upstreamfrom the predicted signal sequence. However, it is likely that thepredicted signal sequence will be capable of directing the secretedprotein to the ER. Nonetheless, the present invention provides themature protein produced by expression of the polynucleotide sequence ofSEQ ID NO:X and/or the polynucleotide sequence contained in the cDNA ofa deposited clone, in a mammalian cell (e.g., COS cells, as describedbelow). These polypeptides, and the polynucleotides encoding suchpolypeptides, are contemplated by the present invention.

Polynucleotide and Polypeptide Variants

The present invention also encompasses variants (e.g., allelic variants,orthologs, etc.) of the polynucleotide sequence disclosed herein in SEQID NO:X, the complementary strand thereto, and/or the cDNA sequencecontained in the deposited clone.

The present invention also encompasses variants of the polypeptidesequence, and/or fragments therein, disclosed in SEQ ID NO:Y, apolypeptide encoded by the polynucleotide sequence in SEQ ID NO:X,and/or a polypeptide encoded by a cDNA in the deposited clone.

“Variant” refers to a polynucleotide or polypeptide differing from thepolynucleotide or polypeptide of the present invention, but retainingessential properties thereof. Generally, variants are overall closelysimilar, and, in many regions, identical to the polynucleotide orpolypeptide of the present invention.

Thus, one aspect of the invention provides an isolated nucleic acidmolecule comprising, or alternatively consisting of, a polynucleotidehaving a nucleotide sequence selected from the group consisting of: (a)a nucleotide sequence encoding a human phosphatase related polypeptidehaving an amino acid sequence as shown in the sequence listing anddescribed in SEQ ID NO:X or the cDNA contained in ATCC deposit No:Z; (b)a nucleotide sequence encoding a mature human phosphatase relatedpolypeptide having the amino acid sequence as shown in the sequencelisting and described in SEQ ID NO:X or the cDNA contained in ATCCdeposit No:Z; (c) a nucleotide sequence encoding a biologically activefragment of a human phosphatase related polypeptide having an amino acidsequence shown in the sequence listing and described in SEQ ID NO:X orthe cDNA contained in ATCC deposit No:Z; (d) a nucleotide sequenceencoding an antigenic fragment of a human phosphatase relatedpolypeptide having an amino acid sequence sown in the sequence listingand described in SEQ ID NO:X or the cDNA contained in ATCC deposit No:Z;(e) a nucleotide sequence encoding a human phosphatase relatedpolypeptide comprising the complete amino acid sequence encoded by ahuman cDNA plasmid contained in SEQ ID NO:X or the cDNA contained inATCC deposit No:Z; (f) a nucleotide sequence encoding a mature humanphosphatase related polypeptide having an amino acid sequence encoded bya human cDNA plasmid contained in SEQ ID NO:X or the cDNA contained inATCC deposit No:Z; (g) a nucleotide sequence encoding a biologicallyactive fragment of a human phosphatase related polypeptide having anamino acid sequence encoded by a human cDNA plasmid contained in SEQ IDNO:X or the cDNA contained in ATCC deposit No:Z; (h) a nucleotidesequence encoding an antigenic fragment of a human phosphatase relatedpolypeptide having an amino acid sequence encoded by a human cDNAplasmid contained in SEQ ID NO:X or the cDNA contained in ATCC depositNo:Z; (I) a nucleotide sequence complimentary to any of the nucleotidesequences in (a), (b), (c), (d), (e), (f), (g), or (h), above.

The present invention is also directed to polynucleotide sequences whichcomprise, or alternatively consist of, a polynucleotide sequence whichis at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to, for example, any of the nucleotide sequences in (a),(b), (c), (d), (e), (f), (g), or (h), above. Polynucleotides encoded bythese nucleic acid molecules are also encompassed by the invention. Inanother embodiment, the invention encompasses nucleic acid moleculeswhich comprise, or alternatively, consist of a polynucleotide whichhybridizes under stringent conditions, or alternatively, under lowerstringency conditions, to a polynucleotide in (a), (b), (c), (d), (e),(f), (g), or (h), above. Polynucleotides which hybridize to thecomplement of these nucleic acid molecules under stringent hybridizationconditions or alternatively, under lower stringency conditions, are alsoencompassed by the invention, as are polypeptides encoded by thesepolypeptides.

Another aspect of the invention provides an isolated nucleic acidmolecule comprising, or alternatively, consisting of, a polynucleotidehaving a nucleotide sequence selected from the group consisting of: (a)a nucleotide sequence encoding a human phosphatase related polypeptidehaving an amino acid sequence as shown in the sequence listing anddescried in Table I; (b) a nucleotide sequence encoding a mature humanphosphatase related polypeptide having the amino acid sequence as shownin the sequence listing and descried in Table I; (c) a nucleotidesequence encoding a biologically active fragment of a human phosphataserelated polypeptide having an amino acid sequence as shown in thesequence listing and descried in Table I; (d) a nucleotide sequenceencoding an antigenic fragment of a human phosphatase relatedpolypeptide having an amino acid sequence as shown in the sequencelisting and described in Table I; (e) a nucleotide sequence encoding ahuman phosphatase related polypeptide comprising the complete amino acidsequence encoded by a human cDNA in a cDNA plasmid contained in the ATCCDeposit and described in Table I; (f) a nucleotide sequence encoding amature human phosphatase related polypeptide having an amino acidsequence encoded by a human cDNA in a cDNA plasmid contained in the ATCCDeposit and described in Table I: (g) a nucleotide sequence encoding abiologically active fragment of a human phosphatase related polypeptidehaving an amino acid sequence encoded by a human cDNA in a cDNA plasmidcontained in the ATCC Deposit and described in Table I; (h) a nucleotidesequence encoding an antigenic fragment of a human phosphatase relatedpolypeptide having an amino acid sequence encoded by a human cDNA in acDNA plasmid contained in the ATCC deposit and described in Table I; (i)a nucleotide sequence complimentary to any of the nucleotide sequencesin (a), (b), (c), (d), (e), (f), (g), or (h) above.

The present invention is also directed to nucleic acid molecules whichcomprise, or alternatively, consist of, a nucleotide sequence which isat least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to, for example, any of the nucleotide sequences in (a), (b),(c), (d), (e), (f), (g), or (h), above.

The present invention encompasses polypeptide sequences which comprise,or alternatively consist of, an amino acid sequence which is at least80%, 98%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identicalto, the following non-limited examples, the polypeptide sequenceidentified as SEQ ID NO:Y, the polypeptide sequence encoded by a cDNAprovided in the deposited clone, and/or polypeptide fragments of any ofthe polypeptides provided herein. Polynucleotides encoded by thesenucleic acid molecules are also encompassed by the invention. In anotherembodiment, the invention encompasses nucleic acid molecules whichcomprise, or alternatively, consist of a polynucleotide which hybridizesunder stringent conditions, or alternatively, under lower stringencyconditions, to a polynucleotide in (a), (b), (c), (d), (e), (f), (g), or(h), above. Polynucleotides which hybridize to the complement of thesenucleic acid molecules under stringent hybridization conditions oralternatively, under lower stringency conditions, are also encompassedby the invention, as are polypeptides encoded by these polypeptides.

The present invention is also directed to polypeptides which comprise,or alternatively consist of, an amino acid sequence which is at least80%, 98%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identicalto, for example, the polypeptide sequence shown in SEQ ID NO:Y, apolypeptide sequence encoded by the nucleotide sequence in SEQ ID NO:X,a polypeptide sequence encoded by the cDNA in cDNA plasmid:Z, and/orpolypeptide fragments of any of these polypeptides (e.g., thosefragments described herein). Polynucleotides which hybridize to thecomplement of the nucleic acid molecules encoding these polypeptidesunder stringent hybridization conditions or alternatively, under lowerstringency conditions, are also encompasses by the present invention, asare the polypeptides encoded by these polynucleotides.

By a nucleic acid having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence of the presentinvention, it is intended that the nucleotide sequence of the nucleicacid is identical to the reference sequence except that the nucleotidesequence may include up to five point mutations per each 100 nucleotidesof the reference nucleotide sequence encoding the polypeptide. In otherwords, to obtain a nucleic acid having a nucleotide sequence at least95% identical to a reference nucleotide sequence, up to 5% of thenucleotides in the reference sequence may be deleted or substituted withanother nucleotide, or a number of nucleotides up to 5% of the totalnucleotides in the reference sequence may be inserted into the referencesequence. The query sequence may be an entire sequence referenced inTable I, the ORF (open reading frame), or any fragment specified asdescribed herein.

As a practical matter, whether any particular nucleic acid molecule orpolypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% nucleotide sequence of the present invention can bedetermined conventionally using known computer programs. A preferredmethod for determining the best overall match between a query sequence(a sequence of the present invention) and a subject sequence, alsoreferred to as a global sequence alignment, can be determined using theCLUSTALW computer program (Thompson, J. D., et al., Nucleic AcidsResearch, 2(22):4673-4680, (1994)), which is based on the algorithm ofHiggins, D. G., et al., Computer Applications in the Biosciences(CABIOS), 8(2): 189-191, (1992). In a sequence alignment the query andsubject sequences are both DNA sequences. An RNA sequence can becompared by converting U's to T's. However, the CLUSTALW algorithmautomatically converts U's to T's when comparing RNA sequences to DNAsequences. The result of said global sequence alignment is in percentidentity. Preferred parameters used in a CLUSTALW alignment of DNAsequences to calculate percent identity via pairwise alignments are:Matrix=IUB, k-tuple=1, Number of Top Diagonals=5, Gap Penalty=3, GapOpen Penalty 10, Gap Extension Penalty=0.1, Scoring Method=Percent,Window Size=5 or the length of the subject nucleotide sequence,whichever is shorter. For multiple alignments, the following CLUSTALWparameters are preferred: Gap Opening Penalty=10; Gap ExtensionParameter=0.05; Gap Separation Penalty Range=8; End Gap SeparationPenalty=Off; % Identity for Alignment Delay=40%; Residue Specific Gaps:Off; Hydrophilic Residue Gap=Off; and Transition Weighting=0. Thepairwise and multple alignment parameters provided for CLUSTALW aboverepresent the default parameters as provided with the AlignX softwareprogram (Vector NTI suite of programs, version 6.0).

The present invention encompasses the application of a manual correctionto the percent identity results, in the instance where the subjectsequence is shorter than the query sequence because of 5′ or 3′deletions, not because of internal deletions. If only the local pairwisepercent identity is required, no manual correction is needed. However, amanual correction may be applied to determine the global percentidentity from a global polynucleotide alignment. Percent identitycalculations based upon global polynucleotide alignments are oftenpreferred since they reflect the percent identity between thepolynucleotide molecules as a whole (i.e., including any polynucleotideoverhangs, not just overlapping regions), as opposed to, only localmatching polynucleotides. Manual corrections for global percent identitydeterminations are required since the CLUSTALW program does not accountfor 5′ and 3′ truncations of the subject sequence when calculatingpercent identity. For subject sequences truncated at the 5′ or 3′ ends,relative to the query sequence, the percent identity is corrected bycalculating the number of bases of the query sequence that are 5′ and 3′of the subject sequence, which are not matched/aligned, as a percent ofthe total bases of the query sequence. Whether a nucleotide ismatched/aligned is determined by results of the CLUSTALW sequencealignment. This percentage is then subtracted from the percent identity,calculated by the above CLUSTALW program using the specified parameters,to arrive at a final percent identity score. This corrected score may beused for the purposes of the present invention. Only bases outside the5′ and 3′ bases of the subject sequence, as displayed by the CLUSTALWalignment, which are not matched/aligned with the query sequence, arecalculated for the purposes of manually adjusting the percent identityscore.

For example, a 90 base subject sequence is aligned to a 100 base querysequence to determine percent identity. The deletions occur at the 5′end of the subject sequence and therefore, the CLUSTALW alignment doesnot show a matched/alignment of the first 10 bases at 5′ end. The 10unpaired bases represent 10% of the sequence (number of bases at the 5′and 3′ ends not matched/total number of bases in the query sequence) so10% is subtracted from the percent identity score calculated by theCLUSTALW program. If the remaining 90 bases were perfectly matched thefinal percent identity would be 90%. In another example, a 90 basesubject sequence is compared with a 100 base query sequence. This timethe deletions are internal deletions so that there are no bases on the5′ or 3′ of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by CLUSTALW is notmanually corrected. Once again, only bases 5′ and 3′ of the subjectsequence which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are required for thepurposes of the present invention.

In addition to the above method of aligning two or more polynucleotideor polypeptide sequences to arrive at a percent identity value for thealigned sequences, it may be desirable in some circumstances to use amodified version of the CLUSTALW algorithm which takes into accountknown structural features of the sequences to be aligned, such as forexample, the SWISS-PROT designations for each sequence. The result ofsuch a modifed CLUSTALW algorithm may provide a more accurate value ofthe percent identity for two polynucleotide or polypeptide sequences.Support for such a modified version of CLUSTALW is provided within theCLUSTALW algorithm and would be readily appreciated to one of skill inthe art of bioinformatics.

The variants may contain alterations in the coding regions, non-codingregions, or both. Especially preferred are polynucleotide variantscontaining alterations which produce silent substitutions, additions, ordeletions, but do not alter the properties or activities of the encodedpolypeptide. Nucleotide variants produced by silent substitutions due tothe degeneracy of the genetic code are preferred. Moreover, variants inwhich 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or addedin any combination are also preferred. Polynucleotide variants can beproduced for a variety of reasons, e.g., to optimize codon expressionfor a particular host (change codons in the mRNA to those preferred by abacterial host such as E. coli).

Naturally occurring variants are called “allelic variants,” and refer toone of several alternate forms of a gene occupying a given locus on achromosome of an organism. (Genes II, Lewin, B., ed., John Wiley & Sons,New York (1985).) These allelic variants can vary at either thepolynucleotide and/or polypeptide level and are included in the presentinvention. Alternatively, non-naturally occurring variants may beproduced by mutagenesis techniques or by direct synthesis.

Using known methods of protein engineering and recombinant DNAtechnology, variants may be generated to improve or alter thecharacteristics of the polypeptides of the present invention. Forinstance, one or more amino acids can be deleted from the N-terminus orC-terminus of the protein without substantial loss of biologicalfunction. The authors of Ron et al., J. Biol. Chem. 268: 2984-2988(1993), reported variant KGF proteins having heparin binding activityeven after deleting 3, 8, or 27 amino-terminal amino acid residues.Similarly, Interferon gamma exhibited up to ten times higher activityafter deleting 8-10 amino acid residues from the carboxy terminus ofthis protein (Dobeli et al., J. Biotechnology 7:199-216 (1988)).

Moreover, ample evidence demonstrates that variants often retain abiological activity similar to that of the naturally occurring protein.For example, Gayle and coworkers (J. Biol. Chem. 268:22105-22111 (1993))conducted extensive mutational analysis of human cytokine IL-1a. Theyused random mutagenesis to generate over 3,500 individual IL-1a mutantsthat averaged 2.5 amino acid changes per variant over the entire lengthof the molecule. Multiple mutations were examined at every possibleamino acid position. The investigators found that “[m]ost of themolecule could be altered with little effect on either [binding orbiological activity].” In fact, only 23 unique amino acid sequences, outof more than 3,500 nucleotide sequences examined, produced a proteinthat significantly differed in activity from wild-type.

Furthermore, even if deleting one or more amino acids from theN-terminus or C-terminus of a polypeptide results in modification orloss of one or more biological functions, other biological activitiesmay still be retained. For example, the ability of a deletion variant toinduce and/or to bind antibodies which recognize the protein will likelybe retained when less than the majority of the residues of the proteinare removed from the N-terminus or C-terminus. Whether a particularpolypeptide lacking N- or C-terminal residues of a protein retains suchimmunogenic activities can readily be determined by routine methodsdescribed herein and otherwise known in the art.

Alternatively, such N-terminus or C-terminus deletions of a polypeptideof the present invention may, in fact, result in a significant increasein one or more of the biological activities of the polypeptide(s). Forexample, biological activity of many polypeptides are governed by thepresence of regulatory domains at either one or both termini. Suchregulatory domains effectively inhibit the biological activity of suchpolypeptides in lieu of an activation event (e.g., binding to a cognateligand or receptor, phosphorylation, proteolytic processing, etc.).Thus, by eliminating the regulatory domain of a polypeptide, thepolypeptide may effectively be rendered biologically active in theabsence of an activation event.

Thus, the invention further includes polypeptide variants that showsubstantial biological activity. Such variants include deletions,insertions, inversions, repeats, and substitutions selected according togeneral rules known in the art so as have little effect on activity. Forexample, guidance concerning how to make phenotypically silent aminoacid substitutions is provided in Bowie et al., Science 247:1306-1310(1990), wherein the authors indicate that there are two main strategiesfor studying the tolerance of an amino acid sequence to change.

The first strategy exploits the tolerance of amino acid substitutions bynatural selection during the process of evolution. By comparing aminoacid sequences in different species, conserved amino acids can beidentified. These conserved amino acids are likely important for proteinfunction. In contrast, the amino acid positions where substitutions havebeen tolerated by natural selection indicates that these positions arenot critical for protein function. Thus, positions tolerating amino acidsubstitution could be modified while still maintaining biologicalactivity of the protein.

The second strategy uses genetic engineering to introduce amino acidchanges at specific positions of a cloned gene to identify regionscritical for protein function. For example, site directed mutagenesis oralanine-scanning mutagenesis (introduction of single alanine mutationsat every residue in the molecule) can be used. (Cunningham and Wells,Science 244:1081-1085 (1989).) The resulting mutant molecules can thenbe tested for biological activity.

As the authors state, these two strategies have revealed that proteinsare surprisingly tolerant of amino acid substitutions. The authorsfurther indicate which amino acid changes are likely to be permissive atcertain amino acid positions in the protein. For example, most buried(within the tertiary structure of the protein) amino acid residuesrequire nonpolar side chains, whereas few features of surface sidechains are generally conserved.

The invention encompasses polypeptides having a lower degree of identitybut having sufficient similarity so as to perform one or more of thesame functions performed by the polypeptide of the present invention.Similarity is determined by conserved amino acid substitution. Suchsubstitutions are those that substitute a given amino acid in apolypeptide by another amino acid of like characteristics (e.g.,chemical properties). According to Cunningham et al above, suchconservative substitutions are likely to be phenotypically silent.Additional guidance concerning which amino acid changes are likely to bephenotypically silent are found in Bowie et al., Science 247:1306-1310(1990).

Tolerated conservative amino acid substitutions of the present inventioninvolve replacement of the aliphatic or hydrophobic amino acids Ala,Val, Leu and Ile; replacement of the hydroxyl residues Ser and Thr;replacement of the acidic residues Asp and Glu; replacement of the amideresidues Asn and Gin, replacement of the basic residues Lys, Arg, andHis; replacement of the aromatic residues Phe, Tyr, and Trp, andreplacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly.

In addition, the present invention also encompasses the conservativesubstitutions provided in Table VII below.

TABLE VII For Amino Acid Code Replace with any of: Alanine A D-Ala, Gly,beta-Ala, L-Cys, D-Cys Arginine R D-Arg, Lys, D-Lys, homo-Arg,D-homo-Arg, Met, Ile, D-Met, D-Ile, Orn, D-Orn Asparagine N D-Asn, Asp,D-Asp, Glu, D-Glu, Gln, D-Gln Aspartic Acid D D-Asp, D-Asn, Asn, Glu,D-Glu, Gln, D-Gln Cysteine C D-Cys, S-Me-Cys, Met, D-Met, Thr, D-ThrGlutamine Q D-Gln, Asn, D-Asn, Glu, D-Glu, Asp, D-Asp Glutamic Acid ED-Glu, D-Asp, Asp, Asn, D-Asn, Gln, D-Gln Glycine G Ala, D-Ala, Pro,D-Pro, β-Ala, Acp Isoleucine I D-Ile, Val, D-Val, Leu, D-Leu, Met, D-MetLeucine L D-Leu, Val, D-Val, Met, D-Met Lysine K D-Lys, Arg, D-Arg,homo-Arg, D-homo-Arg, Met, D-Met, Ile, D-Ile, Orn, D-Orn Methionine MD-Met, S-Me-Cys, Ile, D-Ile, Leu, D-Leu, Val, D-Val Phenylalanine FD-Phe, Tyr, D-Thr, L-Dopa, His, D-His, Trp, D-Trp, Trans-3,4, or5-phenylproline, cis-3,4, or 5-phenylproline Proline P D-Pro,L-1-thioazolidine-4-carboxylic acid, D- or L-1-oxazolidine-4-carboxylicacid Serine S D-Ser, Thr, D-Thr, allo-Thr, Met, D-Met, Met(O), D-Met(O),L-Cys, D-Cys Threonine T D-Thr, Ser, D-Ser, allo-Thr, Met, D-Met,Met(O), D-Met(O), Val, D-Val Tyrosine Y D-Tyr, Phe, D-Phe, L-Dopa, His,D-His Valine V D-Val, Leu, D-Leu, Ile, D-Ile, Met, D-Met

Aside from the uses described above, such amino acid substitutions mayalso increase protein or peptide stability. The invention encompassesamino acid substitutions that contain, for example, one or morenon-peptide bonds (which replace the peptide bonds) in the protein orpeptide sequence. Also included are substitutions that include aminoacid residues other than naturally occurring L-amino acids, e.g.,D-amino acids or non-naturally occurring or synthetic amino acids, e.g.,β or γ amino acids.

Both identity and similarity can be readily calculated by reference tothe following publications: Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing:Informatics and Genome Projects, Smith, D. W., ed., Academic Press, NewYork, 1993; Informatics Computer Analysis of Sequence Data, Part 1,Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey,1994; Sequence Analysis in Molecular Biology, von Heinje, G., AcademicPress, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux,J., eds., M Stockton Press, New York, 1991.

In addition, the present invention also encompasses substitution ofamino acids based upon the probability of an amino acid substitutionresulting in conservation of function. Such probabilities are determinedby aligning multiple genes with related function and assessing therelative penalty of each substitution to proper gene function. Suchprobabilities are often described in a matrix and are used by somealgorithms (e.g., BLAST, CLUSTALW, GAP, etc.) in calculating percentsimilarity wherein similarity refers to the degree by which one aminoacid may substitute for another amino acid without lose of function. Anexample of such a matrix is the PAM250 or BLOSUM62 matrix.

Aside from the canonical chemically conservative substitutionsreferenced above, the invention also encompasses substitutions which aretypically not classified as conservative, but that may be chemicallyconservative under certain circumstances. Analysis of enzymaticcatalysis for proteases, for example, has shown that certain amino acidswithin the active site of some enzymes may have highly perturbed pKa'sdue to the unique microenvironment of the active site. Such perturbedpKa's could enable some amino acids to substitute for other amino acidswhile conserving enzymatic structure and function. Examples of aminoacids that are known to have amino acids with perturbed pKa's are theGlu-35 residue of Lysozyme, the Ile-16 residue of Chymotrypsin, theHis-159 residue of Papain, etc. The conservation of function relates toeither anomalous protonation or anomalous deprotonation of such aminoacids, relative to their canonical, non-perturbed pKa. The pKaperturbation may enable these amino acids to actively participate ingeneral acid-base catalysis due to the unique ionization environmentwithin the enzyme active site. Thus, substituting an amino acid capableof serving as either a general acid or general base within themicroenvironment of an enzyme active site or cavity, as may be the case,in the same or similar capacity as the wild-type amino acid, wouldeffectively serve as a conservative amino substitution.

Besides conservative amino acid substitution, variants of the presentinvention include, but are not limited to, the following: (i)substitutions with one or more of the non-conserved amino acid residues,where the substituted amino acid residues may or may not be one encodedby the genetic code, or (ii) substitution with one or more of amino acidresidues having a substituent group, or (iii) fusion of the maturepolypeptide with another compound, such as a compound to increase thestability and/or solubility of the polypeptide (for example,polyethylene glycol), or (iv) fusion of the polypeptide with additionalamino acids, such as, for example, an IgG Fc fusion region peptide, orleader or secretory sequence, or a sequence facilitating purification.Such variant polypeptides are deemed to be within the scope of thoseskilled in the art from the teachings herein.

For example, polypeptide variants containing amino acid substitutions ofcharged amino acids with other charged or neutral amino acids mayproduce proteins with improved characteristics, such as lessaggregation. Aggregation of pharmaceutical formulations both reducesactivity and increases clearance due to the aggregate's immunogenicactivity. (Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967);Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev.Therapeutic Drug Carrier Systems 10:307-377 (1993).)

Moreover, the invention further includes polypeptide variants createdthrough the application of molecular evolution (“DNA Shuffling”)methodology to the polynucleotide disclosed as SEQ ID NO:X, the sequenceof the clone submitted in a deposit, and/or the cDNA encoding thepolypeptide disclosed as SEQ ID NO:Y. Such DNA Shuffling technology isknown in the art and more particularly described elsewhere herein (e.g.,WPC, Stemmer, PNAS, 91:10747, (1994)), and in the Examples providedherein).

A further embodiment of the invention relates to a polypeptide whichcomprises the amino acid sequence of the present invention having anamino acid sequence which contains at least one amino acid substitution,but not more than 50 amino acid substitutions, even more preferably, notmore than 40 amino acid substitutions, still more preferably, not morethan 30 amino acid substitutions, and still even more preferably, notmore than 20 amino acid substitutions. Of course, in order ofever-increasing preference, it is highly preferable for a peptide orpolypeptide to have an amino acid sequence which comprises the aminoacid sequence of the present invention, which contains at least one, butnot more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions.In specific embodiments, the number of additions, substitutions, and/ordeletions in the amino acid sequence of the present invention orfragments thereof (e.g., the mature form and/or other fragmentsdescribed herein), is 1-5, 5-10, 5-25, 5-50, 10-50 or 50-150,conservative amino acid substitutions are preferable.

Polynucleotide and Polypeptide Fragments

The present invention is directed to polynucleotide fragments of thepolynucleotides of the invention, in addition to polypeptides encodedtherein by said polynucleotides and/or fragments.

In the present invention, a “polynucleotide fragment” refers to a shortpolynucleotide having a nucleic acid sequence which: is a portion ofthat contained in a deposited clone, or encoding the polypeptide encodedby the cDNA in a deposited clone; is a portion of that shown in SEQ IDNO:X or the complementary strand thereto, or is a portion of apolynucleotide sequence encoding the polypeptide of SEQ ID NO:Y. Thenucleotide fragments of the invention are preferably at least about 15nt, and more preferably at least about 20 nt, still more preferably atleast about 30 nt, and even more preferably, at least about 40 nt, atleast about 50 nt, at least about 75 nt, or at least about 150 nt inlength. A fragment “at least 20 nt in length,” for example, is intendedto include 20 or more contiguous bases from the cDNA sequence containedin a deposited clone or the nucleotide sequence shown in SEQ ID NO:X. Inthis context “about” includes the particularly recited value, a valuelarger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at eitherterminus, or at both termini. These nucleotide fragments have uses thatinclude, but are not limited to, as diagnostic probes and primers asdiscussed herein. Of course, larger fragments (e.g., 50, 150, 500, 600,2000 nucleotides) are preferred.

Moreover, representative examples of polynucleotide fragments of theinvention, include, for example, fragments comprising, or alternativelyconsisting of, a sequence from about nucleotide number 1-50, 51-100,101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-450, 451-500,501-550, 551-600, 651-700, 701-750, 751-800, 800-850, 851-900, 901-950,951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200, I201-1250,1251-1300, 1301-1350, 1351-1400, 1401-1450, 1451-1500, 1501-1550,1551-1600, 1601-1650, 1651-1700, 1701-1750, 1751-1800, 1801-1850,1851-1900, 1901-1950, 1951-2000, or 2001 to the end of SEQ ID NO:X, orthe complementary strand thereto, or the cDNA contained in a depositedclone. In this context “about” includes the particularly recited ranges,and ranges larger or smaller by several (5, 4, 3, 2, or 1) nucleotides,at either terminus or at both termini. Preferably, these fragmentsencode a polypeptide which has biological activity. More preferably,these polynucleotides can be used as probes or primers as discussedherein. Also encompassed by the present invention are polynucleotideswhich hybridize to these nucleic acid molecules under stringenthybridization conditions or lower stringency conditions, as are thepolypeptides encoded by these polynucleotides.

In the present invention, a “polypeptide fragment” refers to an aminoacid sequence which is a portion of that contained in SEQ ID NO:Y orencoded by the cDNA contained in a deposited clone. Protein(polypeptide) fragments may be “free-standing,” or comprised within alarger polypeptide of which the fragment forms a part or region, mostpreferably as a single continuous region. Representative examples ofpolypeptide fragments of the invention, include, for example, fragmentscomprising, or alternatively consisting of, from about amino acid number1-20, 21-40, 41-60, 61-80, 81-100, 102-120, 121-140, 141-160, or 161 tothe end of the coding region. Moreover, polypeptide fragments can beabout 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150amino acids in length. In this context “about” includes the particularlyrecited ranges or values, and ranges or values larger or smaller byseveral (5, 4, 3, 2, or 1) amino acids, at either extreme or at bothextremes. Polynucleotides encoding these polypeptides are alsoencompassed by the invention.

Preferred polypeptide fragments include the full-length protein. Furtherpreferred polypeptide fragments include the full-length protein having acontinuous series of deleted residues from the amino or the carboxyterminus, or both. For example, any number of amino acids, ranging from1-60, can be deleted from the amino terminus of the full-lengthpolypeptide. Similarly, any number of amino acids, ranging from 1-30,can be deleted from the carboxy terminus of the full-length protein.Furthermore, any combination of the above amino and carboxy terminusdeletions are preferred. Similarly, polynucleotides encoding thesepolypeptide fragments are also preferred.

Also preferred are polypeptide and polynucleotide fragmentscharacterized by structural or functional domains, such as fragmentsthat comprise alpha-helix and alpha-helix forming regions, beta-sheetand beta-sheet-forming regions, turn and turn-forming regions, coil andcoil-forming regions, hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, flexible regions,surface-forming regions, substrate binding region, and high antigenicindex regions. Polypeptide fragments of SEQ ID NO:Y falling withinconserved domains are specifically contemplated by the presentinvention. Moreover, polynucleotides encoding these domains are alsocontemplated.

Other preferred polypeptide fragments are biologically active fragments.Biologically active fragments are those exhibiting activity similar, butnot necessarily identical, to an activity of the polypeptide of thepresent invention. The biological activity of the fragments may includean improved desired activity, or a decreased undesirable activity.Polynucleotides encoding these polypeptide fragments are alsoencompassed by the invention.

In a preferred embodiment, the functional activity displayed by apolypeptide encoded by a polynucleotide fragment of the invention may beone or more biological activities typically associated with thefull-length polypeptide of the invention. Illustrative of thesebiological activities includes the fragments ability to bind to at leastone of the same antibodies which bind to the full-length protein, thefragments ability to interact with at lease one of the same proteinswhich bind to the full-length, the fragments ability to elicit at leastone of the same immune responses as the full-length protein (i.e., tocause the immune system to create antibodies specific to the sameepitope, etc.), the fragments ability to bind to at least one of thesame polynucleotides as the full-length protein, the fragments abilityto bind to a receptor of the full-length protein, the fragments abilityto bind to a ligand of the full-length protein, and the fragmentsability to multimerize with the full-length protein. However, theskilled artisan would appreciate that some fragments may have biologicalactivities which are desirable and directly inapposite to the biologicalactivity of the full-length protein. The functional activity ofpolypeptides of the invention, including fragments, variants,derivatives, and analogs thereof can be determined by numerous methodsavailable to the skilled artisan, some of which are described elsewhereherein.

The present invention encompasses polypeptides comprising, oralternatively consisting of, an epitope of the polypeptide having anamino acid sequence of SEQ ID NO:Y, or an epitope of the polypeptidesequence encoded by a polynucleotide sequence contained in ATCC depositNo. Z or encoded by a polynucleotide that hybridizes to the complementof the sequence of SEQ ID NO:X or contained in ATCC deposit No. Z understringent hybridization conditions or lower stringency hybridizationconditions as defined supra. The present invention further encompassespolynucleotide sequences encoding an epitope of a polypeptide sequenceof the invention (such as, for example, the sequence disclosed in SEQ IDNO:1), polynucleotide sequences of the complementary strand of apolynucleotide sequence encoding an epitope of the invention, andpolynucleotide sequences which hybridize to the complementary strandunder stringent hybridization conditions or lower stringencyhybridization conditions defined supra.

The term “epitopes,” as used herein, refers to portions of a polypeptidehaving antigenic or immunogenic activity in an animal, preferably amammal, and most preferably in a human. In a preferred embodiment, thepresent invention encompasses a polypeptide comprising an epitope, aswell as the polynucleotide encoding this polypeptide. An “immunogenicepitope,” as used herein, is defined as a portion of a protein thatelicits an antibody response in an animal, as determined by any methodknown in the art, for example, by the methods for generating antibodiesdescribed infra. (See, for example, Geysen et al., Proc. Natl. Acad.Sci. USA 81:3998-4002 (1983)). The term “antigenic epitope,” as usedherein, is defined as a portion of a protein to which an antibody canimmunospecifically bind its antigen as determined by any method wellknown in the art, for example, by the immunoassays described herein.Immunospecific binding excludes non-specific binding but does notnecessarily exclude cross-reactivity with other antigens. Antigenicepitopes need not necessarily be immunogenic.

Fragments which function as epitopes may be produced by any conventionalmeans. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA 82:5131-5135(1985), further described in U.S. Pat. No. 4,631,211).

In the present invention, antigenic epitopes preferably contain asequence of at least 4, at least 5, at least 6, at least 7, morepreferably at least 8, at least 9, at least 10, at least 11, at least12, at least 13, at least 14, at least 15, at least 20, at least 25, atleast 30, at least 40, at least 50, and, most preferably, between about15 to about 30 amino acids. Preferred polypeptides comprisingimmunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acidresidues in length. Additional non-exclusive preferred antigenicepitopes include the antigenic epitopes disclosed herein, as well asportions thereof. Antigenic epitopes are useful, for example, to raiseantibodies, including monoclonal antibodies, that specifically bind theepitope. Preferred antigenic epitopes include the antigenic epitopesdisclosed herein, as well as any combination of two, three, four, fiveor more of these antigenic epitopes. Antigenic epitopes can be used asthe target molecules in immunoassays. (See, for instance, Wilson et al.,Cell 37:767-778 (1984); Sutcliffe et al., Science 219:660-666 (1983)).

Similarly, immunogenic epitopes can be used, for example, to induceantibodies according to methods well known in the art. (See, forinstance, Sutcliffe et al., supra; Wilson et al., supra; Chow et al.,Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al., J. Gen. Virol.66:2347-2354 (1985). Preferred immunogenic epitopes include theimmunogenic epitopes disclosed herein, as well as any combination oftwo, three, four, five or more of these immunogenic epitopes. Thepolypeptides comprising one or more immunogenic epitopes may bepresented for eliciting an antibody response together with a carrierprotein, such as an albumin, to an animal system (such as rabbit ormouse), or, if the polypeptide is of sufficient length (at least about25 amino acids), the polypeptide may be presented without a carrier.However, immunogenic epitopes comprising as few as 8 to 10 amino acidshave been shown to be sufficient to raise antibodies capable of bindingto, at the very least, linear epitopes in a denatured polypeptide (e.g.,in Western blotting).

Epitope-bearing polypeptides of the present invention may be used toinduce antibodies according to methods well known in the art including,but not limited to, in vivo immunization, in vitro immunization, andphage display methods. See, e.g., Sutcliffe et al., supra; Wilson etal., supra, and Bittle et al., J. Gen. Virol., 66:2347-2354 (1985). Ifin vivo immunization is used, animals may be immunized with freepeptide; however, anti-peptide antibody titer may be boosted by couplingthe peptide to a macromolecular carrier, such as keyhole limpethemacyanin (KLH) or tetanus toxoid. For instance, peptides containingcysteine residues may be coupled to a carrier using a linker such asmaleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptidesmay be coupled to carriers using a more general linking agent such asglutaraldehyde. Animals such as rabbits, rats and mice are immunizedwith either free or carrier-coupled peptides, for instance, byintraperitoneal and/or intradermal injection of emulsions containingabout 100 μg of peptide or carrier protein and Freund's adjuvant or anyother adjuvant known for stimulating an immune response. Several boosterinjections may be needed, for instance, at intervals of about two weeks,to provide a useful titer of anti-peptide antibody which can bedetected, for example, by ELISA assay using free peptide adsorbed to asolid surface. The titer of anti-peptide antibodies in serum from animmunized animal may be increased by selection of anti-peptideantibodies, for instance, by adsorption to the peptide on a solidsupport and elution of the selected antibodies according to methods wellknown in the art.

As one of skill in the art will appreciate, and as discussed above, thepolypeptides of the present invention comprising an immunogenic orantigenic epitope can be fused to other polypeptide sequences. Forexample, the polypeptides of the present invention may be fused with theconstant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portionsthereof (CH1, CH2, CH3, or any combination thereof and portions thereof)resulting in chimeric polypeptides. Such fusion proteins may facilitatepurification and may increase half-life in vivo. This has been shown forchimeric proteins consisting of the first two domains of the humanCD4-polypeptide and various domains of the constant regions of the heavyor light chains of mammalian immunoglobulins. See, e.g., EP 394,827;Traunecker et al., Nature, 331:84-86 (1988). Enhanced delivery of anantigen across the epithelial barrier to the immune system has beendemonstrated for antigens (e.g., insulin) conjugated to an FcRn bindingpartner such as IgG or Fc fragments (see, e.g., PCT Publications WO96/22024 and WO 99/04813). IgG Fusion proteins that have adisulfide-linked dimeric structure due to the IgG portion disulfidebonds have also been found to be more efficient in binding andneutralizing other molecules than monomeric polypeptides or fragmentsthereof alone. See, e.g., Fountoulakis et al., J. Biochem.,270:3958-3964 (1995). Nucleic acids encoding the above epitopes can alsobe recombined with a gene of interest as an epitope tag (e.g., thehemagglutinin (“HA”) tag or flag tag) to aid in detection andpurification of the expressed polypeptide. For example, a systemdescribed by Janknecht et al. allows for the ready purification ofnon-denatured fusion proteins expressed in human cell lines (Janknechtet al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-897). In this system,the gene of interest is subcloned into a vaccinia recombination plasmidsuch that the open reading frame of the gene is translationally fused toan amino-terminal tag consisting of six histidine residues. The tagserves as a matrix binding domain for the fusion protein. Extracts fromcells infected with the recombinant vaccinia virus are loaded onto Ni2+nitriloacetic acid-agarose column and histidine-tagged proteins can beselectively eluted with imidazole-containing buffers.

Additional fusion proteins of the invention may be generated through thetechniques of gene-shuffling, motif-shuffling, exon-shuffling, and/orcodon-shuffling (collectively referred to as “DNA shuffling”). DNAshuffling may be employed to modulate the activities of polypeptides ofthe invention, such methods can be used to generate polypeptides withaltered activity, as well as agonists and antagonists of thepolypeptides. See, generally, U.S. Pat. Nos. 5,605,793; 5,811,238;5,830,721; 5,834,252; and 5,837,458, and Patten et al., Curr. OpinionBiotechnol. 8:724-33 (1997); Harayama, Trends Biotechnol. 16(2):76-82(1998); Hansson, et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzoand Blasco, Biotechniques 24(2):308-13 (1998) (each of these patents andpublications are hereby incorporated by reference in its entirety). Inone embodiment, alteration of polynucleotides corresponding to SEQ IDNO:X and the polypeptides encoded by these polynucleotides may beachieved by DNA shuffling. DNA shuffling involves the assembly of two ormore DNA segments by homologous or site-specific recombination togenerate variation in the polynucleotide sequence. In anotherembodiment, polynucleotides of the invention, or the encodedpolypeptides, may be altered by being subjected to random mutagenesis byerror-prone PCR, random nucleotide insertion or other methods prior torecombination. In another embodiment, one or more components, motifs,sections, parts, domains, fragments, etc., of a polynucleotide encodinga polypeptide of the invention may be recombined with one or morecomponents, motifs, sections, parts, domains, fragments, etc. of one ormore heterologous molecules.

Antibodies

Further polypeptides of the invention relate to antibodies and T-cellantigen receptors (TCR) which immunospecifically bind a polypeptide,polypeptide fragment, or variant of SEQ ID NO:Y, and/or an epitope, ofthe present invention (as determined by immunoassays well known in theart for assaying specific antibody-antigen binding). Antibodies of theinvention include, but are not limited to, polyclonal, monoclonal,monovalent, bispecific, heteroconjugate, multispecific, human, humanizedor chimeric antibodies, single chain antibodies, Fab fragments, F(ab′)fragments, fragments produced by a Fab expression library,anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodiesto antibodies of the invention), and epitope-binding fragments of any ofthe above. The term “antibody,” as used herein, refers to immunoglobulinmolecules and immunologically active portions of immunoglobulinmolecules, i.e., molecules that contain an antigen binding site thatimmunospecifically binds an antigen. The immunoglobulin molecules of theinvention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY),class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass ofimmunoglobulin molecule. Moreover, the term “antibody” (Ab) or“monoclonal antibody” (Mab) is meant to include intact molecules, aswell as, antibody fragments (such as, for example, Fab and F(ab′)2fragments) which are capable of specifically binding to protein. Fab andF(ab′)2 fragments lack the Fc fragment of intact antibody, clear morerapidly from the circulation of the animal or plant, and may have lessnon-specific tissue binding than an intact antibody (Wahl et al., J.Nucl. Med. 24:316-325 (1983)). Thus, these fragments are preferred, aswell as the products of a FAB or other immunoglobulin expressionlibrary. Moreover, antibodies of the present invention include chimeric,single chain, and humanized antibodies.

Most preferably the antibodies are human antigen-binding antibodyfragments of the present invention and include, but are not limited to,Fab, Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv), single-chainantibodies, disulfide-linked Fvs (sdFv) and fragments comprising eithera VL or VH domain. Antigen-binding antibody fragments, includingsingle-chain antibodies, may comprise the variable region(s) alone or incombination with the entirety or a portion of the following: hingeregion, CH1, CH2, and CH3 domains. Also included in the invention areantigen-binding fragments also comprising any combination of variableregion(s) with a hinge region, CH1, CH2, and CH3 domains. The antibodiesof the invention may be from any animal origin including birds andmammals. Preferably, the antibodies are human, murine (e.g., mouse andrat), donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken.As used herein, “human” antibodies include antibodies having the aminoacid sequence of a human immunoglobulin and include antibodies isolatedfrom human immunoglobulin libraries or from animals transgenic for oneor more human immunoglobulin and that do not express endogenousimmunoglobulins, as described infra and, for example in, U.S. Pat. No.5,939,598 by Kucherlapati et al.

The antibodies of the present invention may be monospecific, bispecific,trispecific or of greater multispecificity. Multispecific antibodies maybe specific for different epitopes of a polypeptide of the presentinvention or may be specific for both a polypeptide of the presentinvention as well as for a heterologous epitope, such as a heterologouspolypeptide or solid support material. See, e.g., PCT publications WO93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J.Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681;4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol.148:1547-1553 (1992).

Antibodies of the present invention may be described or specified interms of the epitope(s) or portion(s) of a polypeptide of the presentinvention which they recognize or specifically bind. The epitope(s) orpolypeptide portion(s) may be specified as described herein, e.g., byN-terminal and C-terminal positions, by size in contiguous amino acidresidues, or listed in the Tables and Figures. Antibodies whichspecifically bind any epitope or polypeptide of the present inventionmay also be excluded. Therefore, the present invention includesantibodies that specifically bind polypeptides of the present invention,and allows for the exclusion of the same.

Antibodies of the present invention may also be described or specifiedin terms of their cross-reactivity. Antibodies that do not bind anyother analog, ortholog, or homologue of a polypeptide of the presentinvention are included. Antibodies that bind polypeptides with at least95%, at least 90%, at least 85%, at least 80%, at least 75%, at least70%, at least 65%, at least 60%, at least 55%, and at least 50% identity(as calculated using methods known in the art and described herein) to apolypeptide of the present invention are also included in the presentinvention. In specific embodiments, antibodies of the present inventioncross-react with murine, rat and/or rabbit homologues of human proteinsand the corresponding epitopes thereof. Antibodies that do not bindpolypeptides with less than 95%, less than 90%, less than 85%, less than80%, less than 75%, less than 70%, less than 65%, less than 60%, lessthan 55%, and less than 50% identity (as calculated using methods knownin the art and described herein) to a polypeptide of the presentinvention are also included in the present invention. In a specificembodiment, the above-described cross-reactivity is with respect to anysingle specific antigenic or immunogenic polypeptide, or combination(s)of 2, 3, 4, 5, or more of the specific antigenic and/or immunogenicpolypeptides disclosed herein. Further included in the present inventionare antibodies which bind polypeptides encoded by polynucleotides whichhybridize to a polynucleotide of the present invention under stringenthybridization conditions (as described herein). Antibodies of thepresent invention may also be described or specified in terms of theirbinding affinity to a polypeptide of the invention. Preferred bindingaffinities include those with a dissociation constant or Kd less than5×10-2 M, 10-2 M, 5×10-3 M, 10-3 M, 5×10-4 M, 10-4 M, 5×10-5 M, 10-5 M,5×10-6 M, 10-6M, 5×10-7 M, 107 M, 5×10-8M, 10-8M, 5×10-9M, 10-9M,5×10-10M, 10-10 M, 5×10-11 M, 10-11 M, 5×10-12 M, 10-12 M, 5×10-13 M,10-13 M, 5×10-14 M, 10-14 M, 5×10-15 M, or 10-15 M.

The invention also provides antibodies that competitively inhibitbinding of an antibody to an epitope of the invention as determined byany method known in the art for determining competitive binding, forexample, the immunoassays described herein. In preferred embodiments,the antibody competitively inhibits binding to the epitope by at least95%, at least 90%, at least 85%, at least 80%, at least 75%, at least70%, at least 60%, or at least 50%.

Antibodies of the present invention may act as agonists or antagonistsof the polypeptides of the present invention. For example, the presentinvention includes antibodies which disrupt the receptor/ligandinteractions with the polypeptides of the invention either partially orfully. Preferably, antibodies of the present invention bind an antigenicepitope disclosed herein, or a portion thereof. The invention featuresboth receptor-specific antibodies and ligand-specific antibodies. Theinvention also features receptor-specific antibodies which do notprevent ligand binding but prevent receptor activation. Receptoractivation (i.e., signaling) may be determined by techniques describedherein or otherwise known in the art. For example, receptor activationcan be determined by detecting the phosphorylation (e.g., tyrosine orserine/threonine) of the receptor or its substrate byimmunoprecipitation followed by western blot analysis (for example, asdescribed supra). In specific embodiments, antibodies are provided thatinhibit ligand activity or receptor activity by at least 95%, at least90%, at least 85%, at least 80%, at least 75%, at least 70%, at least60%, or at least 50% of the activity in absence of the antibody.

The invention also features receptor-specific antibodies which bothprevent ligand binding and receptor activation as well as antibodiesthat recognize the receptor-ligand complex, and, preferably, do notspecifically recognize the unbound receptor or the unbound ligand.Likewise, included in the invention are neutralizing antibodies whichbind the ligand and prevent binding of the ligand to the receptor, aswell as antibodies which bind the ligand, thereby preventing receptoractivation, but do not prevent the ligand from binding the receptor.Further included in the invention are antibodies which activate thereceptor. These antibodies may act as receptor agonists, i.e.,potentiate or activate either all or a subset of the biologicalactivities of the ligand-mediated receptor activation, for example, byinducing dimerization of the receptor. The antibodies may be specifiedas agonists, antagonists or inverse agonists for biological activitiescomprising the specific biological activities of the peptides of theinvention disclosed herein. The above antibody agonists can be madeusing methods known in the art. See, e.g., PCT publication WO 96/40281;U.S. Pat. No. 5,811,097; Deng et al., Blood 92(6):1981-1988 (1998); Chenet al., Cancer Res. 58(16):3668-3678 (1998); Harrop et al., J. Immunol.161(4):1786-1794 (1998); Zhu et al., Cancer Res. 58(15):3209-3214(1998); Yoon et al., J. Immunol. 160(7):3170-3179 (1998); Prat et al.,J. Cell. Sci. 111(Pt2):237-247 (1998); Pitard et al., J. Immunol.Methods 205(2):177-190 (1997); Liautard et al., Cytokine 9(4):233-241(1997); Carlson et al., J. Biol. Chem. 272(17):11295-11301 (1997);Taryman et al., Neuron 14(4):755-762 (1995); Muller et al., Structure6(9):1153-1167 (1998); Bartunek et al., Cytokine 8(1):14-20 (1996)(which are all incorporated by reference herein in their entireties).

Antibodies of the present invention may be used, for example, but notlimited to, to purify, detect, and target the polypeptides of thepresent invention, including both in vitro and in vivo diagnostic andtherapeutic methods. For example, the antibodies have use inimmunoassays for qualitatively and quantitatively measuring levels ofthe polypeptides of the present invention in biological samples. See,e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold SpringHarbor Laboratory Press, 2nd ed. 1988) (incorporated by reference hereinin its entirety).

As discussed in more detail below, the antibodies of the presentinvention may be used either alone or in combination with othercompositions. The antibodies may further be recombinantly fused to aheterologous polypeptide at the N- or C-terminus or chemicallyconjugated (including covalently and non-covalently conjugations) topolypeptides or other compositions. For example, antibodies of thepresent invention may be recombinantly fused or conjugated to moleculesuseful as labels in detection assays and effector molecules such asheterologous polypeptides, drugs, radionucleotides, or toxins. See,e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat.No. 5,314,995; and EP 396,387.

The antibodies of the invention include derivatives that are modified,i.e., by the covalent attachment of any type of molecule to the antibodysuch that covalent attachment does not prevent the antibody fromgenerating an anti-idiotypic response. For example, but not by way oflimitation, the antibody derivatives include antibodies that have beenmodified, e.g., by glycosylation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications may be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Additionally, the derivative may contain one or more non-classicalamino acids.

The antibodies of the present invention may be generated by any suitablemethod known in the art.

The antibodies of the present invention may comprise polyclonalantibodies. Methods of preparing polyclonal antibodies are known to theskilled artisan (Harlow, et al., Antibodies: A Laboratory Manual, (Coldspring Harbor Laboratory Press, 2^(nd) ed. (1988), which is herebyincorporated herein by reference in its entirety). For example, apolypeptide of the invention can be administered to various host animalsincluding, but not limited to, rabbits, mice, rats, etc. to induce theproduction of sera containing polyclonal antibodies specific for theantigen. The administration of the polypeptides of the present inventionmay entail one or more injections of an immunizing agent and, ifdesired, an adjuvant. Various adjuvants may be used to increase theimmunological response, depending on the host species, and include butare not limited to, Freund's (complete and incomplete), mineral gelssuch as aluminum hydroxide, surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and corynebacteriumparvum. Such adjuvants are also well known in the art. For the purposesof the invention, “immunizing agent” may be defined as a polypeptide ofthe invention, including fragments, variants, and/or derivativesthereof, in addition to fusions with heterologous polypeptides and otherforms of the polypeptides described herein.

Typically, the immunizing agent and/or adjuvant will be injected in themammal by multiple subcutaneous or intraperitoneal injections, thoughthey may also be given intramuscularly, and/or through IV). Theimmunizing agent may include polypeptides of the present invention or afusion protein or variants thereof. Depending upon the nature of thepolypeptides (i.e., percent hydrophobicity, percent hydrophilicity,stability, net charge, isoelectric point etc.), it may be useful toconjugate the immunizing agent to a protein known to be immunogenic inthe mammal being immunized. Such conjugation includes either chemicalconjugation by derivitizing active chemical functional groups to boththe polypeptide of the present invention and the immunogenic proteinsuch that a covalent bond is formed, or through fusion-protein basedmethodology, or other methods known to the skilled artisan. Examples ofsuch immunogenic proteins include, but are not limited to keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsininhibitor. Various adjuvants may be used to increase the immunologicalresponse, depending on the host species, including but not limited toFreund's (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,dinitrophenol, and potentially useful human adjuvants such as BCG(bacille Calmette-Guerin) and Corynebacterium parvum. Additionalexamples of adjuvants which may be employed includes the MPL-TDMadjuvant (monophosphoryl lipid A, synthetic trehalose dicorynomycolate).The immunization protocol may be selected by one skilled in the artwithout undue experimentation.

The antibodies of the present invention may comprise monoclonalantibodies. Monoclonal antibodies may be prepared using hybridomamethods, such as those described by Kohler and Milstein, Nature, 256:495(1975) and U.S. Pat. No. 4,376,110, by Harlow, et al., Antibodies: ALaboratory Manual, (Cold spring Harbor Laboratory Press, 2^(nd) ed.(1988), by Hammerling, et al., Monoclonal Antibodies and T-CellHybridomas (Elsevier, N.Y., (1981)), or other methods known to theartisan. Other examples of methods which may be employed for producingmonoclonal antibodies includes, but are not limited to, the human B-cellhybridoma technique (Kosbor et al., 1983, Immunology Today 4:72; Cole etal., 1983, Proc. Natl. Acad. Sci. USA 80:2026-2030), and theEBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies AndCancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such antibodies may beof any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and anysubclass thereof. The hybridoma producing the mAb of this invention maybe cultivated in vitro or in vivo. Production of high titers of mAbs invivo makes this the presently preferred method of production.

In a hybridoma method, a mouse, a humanized mouse, a mouse with a humanimmune system, hamster, or other appropriate host animal, is typicallyimmunized with an immunizing agent to elicit lymphocytes that produce orare capable of producing antibodies that will specifically bind to theimmunizing agent. Alternatively, the lymphocytes may be immunized invitro.

The immunizing agent will typically include polypeptides of the presentinvention or a fusion protein thereof. Generally, either peripheralblood lymphocytes (“PBLs”) are used if cells of human origin aredesired, or spleen cells or lymph node cells are used if non-humanmammalian sources are desired. The lymphocytes are then fused with animmortalized cell line using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, Academic Press, (1986), pp.59-103). Immortalized cell lines are usually transformed mammaliancells, particularly myeloma cells of rodent, bovine and human origin.Usually, rat or mouse myeloma cell lines are employed. The hybridomacells may be cultured in a suitable culture medium that preferablycontains one or more substances that inhibit the growth or survival ofthe unfused, immortalized cells. For example, if the parental cells lackthe enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT orHPRT), the culture medium for the hybridomas typically will includehypoxanthine, aminopterin, and thymidine (“HAT medium”), whichsubstances prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. As inferred throughout the specification,human myeloma and mouse-human heteromyeloma cell lines also have beendescribed for the production of human monoclonal antibodies (Kozbor, J.Immunol., 133:3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, Marcel Dekker, Inc., New York,(1987) pp. 51-63).

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against thepolypeptides of the present invention. Preferably, the bindingspecificity of monoclonal antibodies produced by the hybridoma cells isdetermined by immunoprecipitation or by an in vitro binding assay, suchas radioimmunoassay (RIA) or enzyme-linked immunoadsorbant assay(ELISA). Such techniques are known in the art and within the skill ofthe artisan. The binding affinity of the monoclonal antibody can, forexample, be determined by the Scatchard analysis of Munson and Pollart,Anal. Biochem., 107:220 (1980).

After the desired hybridoma cells are identified, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, supra). Suitable culture media for this purpose include, forexample, Dulbecco's Modified Eagle's Medium and RPMI-1640.Alternatively, the hybridoma cells may be grown in vivo as ascites in amammal.

The monoclonal antibodies secreted by the subclones may be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-sepharose, hydroxyapatite chromatography, gel exclusionchromatography, gel electrophoresis, dialysis, or affinitychromatography.

The skilled artisan would acknowledge that a variety of methods exist inthe art for the production of monoclonal antibodies and thus, theinvention is not limited to their sole production in hydridomas. Forexample, the monoclonal antibodies may be made by recombinant DNAmethods, such as those described in U.S. Pat. No. 4,816,567. In thiscontext, the term “monoclonal antibody” refers to an antibody derivedfrom a single eukaryotic, phage, or prokaryotic clone. The DNA encodingthe monoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies, or such chains from human,humanized, or other sources). The hydridoma cells of the invention serveas a preferred source of such DNA. Once isolated, the DNA may be placedinto expression vectors, which are then transformed into host cells suchas Simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cellsthat do not otherwise produce immunoglobulin protein, to obtain thesynthesis of monoclonal antibodies in the recombinant host cells. TheDNA also may be modified, for example, by substituting the codingsequence for human heavy and light chain constant domains in place ofthe homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison etal, supra) or by covalently joining to the immunoglobulin codingsequence all or part of the coding sequence for a non-immunoglobulinpolypeptide. Such a non-immunoglobulin polypeptide can be substitutedfor the constant domains of an antibody of the invention, or can besubstituted for the variable domains of one antigen-combining site of anantibody of the invention to create a chimeric bivalent antibody.

The antibodies may be monovalent antibodies. Methods for preparingmonovalent antibodies are well known in the art. For example, one methodinvolves recombinant expression of immunoglobulin light chain andmodified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy chain crosslinking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent crosslinking.

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly, Fabfragments, can be accomplished using routine techniques known in theart. Monoclonal antibodies can be prepared using a wide variety oftechniques known in the art including the use of hybridoma, recombinant,and phage display technologies, or a combination thereof. For example,monoclonal antibodies can be produced using hybridoma techniquesincluding those known in the art and taught, for example, in Harlow etal., Antibodies: A Laboratory Manual, (Cold Spring Harbor LaboratoryPress, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies andT-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) (said referencesincorporated by reference in their entireties). The term “monoclonalantibody” as used herein is not limited to antibodies produced throughhybridoma technology. The term “monoclonal antibody” refers to anantibody that is derived from a single clone, including any eukaryotic,prokaryotic, or phage clone, and not the method by which it is produced.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art and arediscussed in detail in the Examples herein. In a non-limiting example,mice can be immunized with a polypeptide of the invention or a cellexpressing such peptide. Once an immune response is detected, e.g.,antibodies specific for the antigen are detected in the mouse serum, themouse spleen is harvested and splenocytes isolated. The splenocytes arethen fused by well-known techniques to any suitable myeloma cells, forexample cells from cell line SP20 available from the ATCC. Hybridomasare selected and cloned by limited dilution. The hybridoma clones arethen assayed by methods known in the art for cells that secreteantibodies capable of binding a polypeptide of the invention. Ascitesfluid, which generally contains high levels of antibodies, can begenerated by immunizing mice with positive hybridoma clones.

Accordingly, the present invention provides methods of generatingmonoclonal antibodies as well as antibodies produced by the method,comprising culturing a hybridoma cell secreting an antibody of theinvention wherein, preferably, the hybridoma is generated by fusingsplenocytes isolated from a mouse immunized with an antigen of theinvention with myeloma cells and then screening the hybridomas resultingfrom the fusion for hybridoma clones that secrete an antibody able tobind a polypeptide of the invention.

Antibody fragments which recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab′)2 fragments of theinvention may be produced by proteolytic cleavage of immunoglobulinmolecules, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)2 fragments). F(ab′)2 fragments contain thevariable region, the light chain constant region and the CH1 domain ofthe heavy chain.

For example, the antibodies of the present invention can also begenerated using various phage display methods known in the art. In phagedisplay methods, functional antibody domains are displayed on thesurface of phage particles which carry the polynucleotide sequencesencoding them. In a particular embodiment, such phage can be utilized todisplay antigen binding domains expressed from a repertoire orcombinatorial antibody library (e.g., human or murine). Phage expressingan antigen binding domain that binds the antigen of interest can beselected or identified with antigen, e.g., using labeled antigen orantigen bound or captured to a solid surface or bead. Phage used inthese methods are typically filamentous phage including fd and M13binding domains expressed from phage with Fab, Fv or disulfidestabilized Fv antibody domains recombinantly fused to either the phagegene III or gene VIII protein. Examples of phage display methods thatcan be used to make the antibodies of the present invention includethose disclosed in Brinkman et al., J. Immunol. Methods 182:41-50(1995); Ames et al., J. Immunol. Methods 184:177-186 (1995);Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al.,Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280(1994); PCT application No. PCT/GB91/01134; PCT publications WO90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409;5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698;5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108;each of which is incorporated herein by reference in its entirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′ and F(ab′)2 fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869(1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al.,Science 240:1041-1043 (1988) (said references incorporated by referencein their entireties). Examples of techniques which can be used toproduce single-chain Fvs and antibodies include those described in U.S.Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993); and Skerra etal., Science 240:1038-1040 (1988).

For some uses, including in vivo use of antibodies in humans and invitro detection assays, it may be preferable to use chimeric, humanized,or human antibodies. A chimeric antibody is a molecule in whichdifferent portions of the antibody are derived from different animalspecies, such as antibodies having a variable region derived from amurine monoclonal antibody and a human immunoglobulin constant region.Methods for producing chimeric antibodies are known in the art. Seee.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214(1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; U.S.Pat. Nos. 5,807,715; 4,816,567; and 4,816,397, which are incorporatedherein by reference in their entirety. Humanized antibodies are antibodymolecules from non-human species antibody that binds the desired antigenhaving one or more complementarity determining regions (CDRs) from thenon-human species and a framework regions from a human immunoglobulinmolecule. Often, framework residues in the human framework regions willbe substituted with the corresponding residue from the CDR donorantibody to alter, preferably improve, antigen binding. These frameworksubstitutions are identified by methods well known in the art, e.g., bymodeling of the interactions of the CDR and framework residues toidentify framework residues important for antigen binding and sequencecomparison to identify unusual framework residues at particularpositions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; Riechmannet al., Nature 332:323 (1988), which are incorporated herein byreference in their entireties.) Antibodies can be humanized using avariety of techniques known in the art including, for example,CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos.5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498(1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994);Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat.No. 5,565,332). Generally, a humanized antibody has one or more aminoacid residues introduced into it from a source that is non-human. Thesenon-human amino acid residues are often referred to as “import”residues, which are typically taken from an “import” variable domain.Humanization can be essentially performed following the methods ofWinter and co-workers (Jones et al., Nature, 321:522-525 (1986);Reichmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science,239:1534-1536 (1988), by substituting rodent CDRs or CDR sequences forthe corresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No.4,816,567), wherein substantially less than an intact human variabledomain has been substituted by the corresponding sequence from anon-human species. In practice, humanized antibodies are typically humanantibodies in which some CDR residues and possible some FR residues aresubstituted from analogous sites in rodent antibodies.

In general, the humanized antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the CDR regions correspond to those of a non-humanimmunoglobulin and all or substantially all of the FR regions are thoseof a human immunoglobulin consensus sequence. The humanized antibodyoptimally also will comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin (Jones etal., Nature, 321:522-525 (1986); Riechmann et al., Nature 332:323-329(1988)1 and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992).

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Human antibodies can be made by a varietyof methods known in the art including phage display methods describedabove using antibody libraries derived from human immunoglobulinsequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCTpublications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO96/34096, WO 96/33735, and WO 91/10741; each of which is incorporatedherein by reference in its entirety. The techniques of cole et al., andBoerder et al., are also available for the preparation of humanmonoclonal antibodies (cole et al., Monoclonal Antibodies and CancerTherapy, Alan R. Riss, (1985); and Boerner et al., J. Immunol.,147(1):86-95, (1991)).

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes may be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring which express human antibodies. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a polypeptide of the invention. Monoclonal antibodiesdirected against the antigen can be obtained from the immunized,transgenic mice using conventional hybridoma technology. The humanimmunoglobulin transgenes harbored by the transgenic mice rearrangeduring B cell differentiation, and subsequently undergo class switchingand somatic mutation. Thus, using such a technique, it is possible toproduce therapeutically useful IgG, IgA, IgM and IgE antibodies. For anoverview of this technology for producing human antibodies, see Lonbergand Huszar, Int. Rev. Immunol. 13:65-93 (1995). For a detaileddiscussion of this technology for producing human antibodies and humanmonoclonal antibodies and protocols for producing such antibodies, see,e.g., PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO96/33735; European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923;5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318;5,885,793; 5,916,771; and 5,939,598, which are incorporated by referenceherein in their entirety. In addition, companies such as Abgenix, Inc.(Freemont, Calif.), Genpharm (San Jose, Calif.), and Medarex, Inc.(Princeton, N.J.) can be engaged to provide human antibodies directedagainst a selected antigen using technology similar to that describedabove.

Similarly, human antibodies can be made by introducing humanimmunoglobulin loci into transgenic animals, e.g., mice in which theendogenous immunoglobulin genes have been partially or completelyinactivated. Upon challenge, human antibody production is observed,which closely resembles that seen in humans in all respects, includinggene rearrangement, assembly, and creation of an antibody repertoire.This approach is described, for example, in U.S. Pat. Nos. 5,545,807;5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,106, and in thefollowing scientific publications: Marks et al., Biotechnol., 10:779-783(1992); Lonberg et al., Nature 368:856-859 (1994); Fishwild et al.,Nature Biotechnol., 14:845-51 (1996); Neuberger, Nature Biotechnol.,14:826 (1996); Lonberg and Huszer, Intern. Rev. Immunol., 13:65-93(1995).

Completely human antibodies which recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al., Bio/technology 12:899-903(1988)).

Further, antibodies to the polypeptides of the invention can, in turn,be utilized to generate anti-idiotype antibodies that “mimic”polypeptides of the invention using techniques well known to thoseskilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444;(1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example,antibodies which bind to and competitively inhibit polypeptidemultimerization and/or binding of a polypeptide of the invention to aligand can be used to generate anti-idiotypes that “mimic” thepolypeptide multimerization and/or binding domain and, as a consequence,bind to and neutralize polypeptide and/or its ligand. Such neutralizinganti-idiotypes or Fab fragments of such anti-idiotypes can be used intherapeutic regimens to neutralize polypeptide ligand. For example, suchanti-idiotypic antibodies can be used to bind a polypeptide of theinvention and/or to bind its ligands/receptors, and thereby block itsbiological activity.

The antibodies of the present invention may be bispecific antibodies.Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present invention, one of the binding specificities maybe directed towards a polypeptide of the present invention, the othermay be for any other antigen, and preferably for a cell-surface protein,receptor, receptor subunit, tissue-specific antigen, virally derivedprotein, virally encoded envelope protein, bacterially derived protein,or bacterial surface protein, etc.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, Nature, 305:537-539 (1983). Because of the random assortmentof immunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of ten different antibody molecules, ofwhich only one has the correct bispecific structure. The purification ofthe correct molecule is usually accomplished by affinity chromatographysteps. Similar procedures are disclosed in WO 93/08829, published 13 May1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transformed into a suitable host organism. Forfurther details of generating bispecific antibodies see, for exampleSuresh et al., Meth. In Enzym., 121:210 (1986).

Heteroconjugate antibodies are also contemplated by the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells (U.S. Pat. No. 4,676,980),and for the treatment of HIV infection (WO 91/00360; WO 92/20373; andEP03089). It is contemplated that the antibodies may be prepared invitro using known methods in synthetic protein chemistry, includingthose involving crosslinking agents. For example, immunotoxins may beconstructed using a disulfide exchange reaction or by forming athioester bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

Polynucleotides Encoding Antibodies

The invention further provides polynucleotides comprising a nucleotidesequence encoding an antibody of the invention and fragments thereof.The invention also encompasses polynucleotides that hybridize understringent or lower stringency hybridization conditions, e.g., as definedsupra, to polynucleotides that encode an antibody, preferably, thatspecifically binds to a polypeptide of the invention, preferably, anantibody that binds to a polypeptide having the amino acid sequence ofSEQ ID NO:Y.

The polynucleotides may be obtained, and the nucleotide sequence of thepolynucleotides determined, by any method known in the art. For example,if the nucleotide sequence of the antibody is known, a polynucleotideencoding the antibody may be assembled from chemically synthesizedoligonucleotides (e.g., as described in Kutmeier et al., BioTechniques17:242 (1994)), which, briefly, involves the synthesis of overlappingoligonucleotides containing portions of the sequence encoding theantibody, annealing and ligating of those oligonucleotides, and thenamplification of the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody may be generatedfrom nucleic acid from a suitable source. If a clone containing anucleic acid encoding a particular antibody is not available, but thesequence of the antibody molecule is known, a nucleic acid encoding theimmunoglobulin may be chemically synthesized or obtained from a suitablesource (e.g., an antibody cDNA library, or a cDNA library generatedfrom, or nucleic acid, preferably poly A+RNA, isolated from, any tissueor cells expressing the antibody, such as hybridoma cells selected toexpress an antibody of the invention) by PCR amplification usingsynthetic primers hybridizable to the 3′ and 5′ ends of the sequence orby cloning using an oligonucleotide probe specific for the particulargene sequence to identify, e.g., a cDNA clone from a cDNA library thatencodes the antibody. Amplified nucleic acids generated by PCR may thenbe cloned into replicable cloning vectors using any method well known inthe art.

Once the nucleotide sequence and corresponding amino acid sequence ofthe antibody is determined, the nucleotide sequence of the antibody maybe manipulated using methods well known in the art for the manipulationof nucleotide sequences, e.g., recombinant DNA techniques, site directedmutagenesis, PCR, etc. (see, for example, the techniques described inSambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel etal., eds., 1998, Current Protocols in Molecular Biology, John Wiley &Sons, NY, which are both incorporated by reference herein in theirentireties), to generate antibodies having a different amino acidsequence, for example to create amino acid substitutions, deletions,and/or insertions.

In a specific embodiment, the amino acid sequence of the heavy and/orlight chain variable domains may be inspected to identify the sequencesof the complementarity determining regions (CDRs) by methods that arewell know in the art, e.g., by comparison to known amino acid sequencesof other heavy and light chain variable regions to determine the regionsof sequence hypervariability. Using routine recombinant DNA techniques,one or more of the CDRs may be inserted within framework regions, e.g.,into human framework regions to humanize a non-human antibody, asdescribed supra. The framework regions may be naturally occurring orconsensus framework regions, and preferably human framework regions(see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479 (1998) for alisting of human framework regions). Preferably, the polynucleotidegenerated by the combination of the framework regions and CDRs encodesan antibody that specifically binds a polypeptide of the invention.Preferably, as discussed supra, one or more amino acid substitutions maybe made within the framework regions, and, preferably, the amino acidsubstitutions improve binding of the antibody to its antigen.Additionally, such methods may be used to make amino acid substitutionsor deletions of one or more variable region cysteine residuesparticipating in an intrachain disulfide bond to generate antibodymolecules lacking one or more intrachain disulfide bonds. Otheralterations to the polynucleotide are encompassed by the presentinvention and within the skill of the art.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984);Neuberger et al., Nature 312:604-608 (1984); Takeda et al., Nature314:452-454 (1985)) by splicing genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Asdescribed supra, a chimeric antibody is a molecule in which differentportions are derived from different animal species, such as those havinga variable region derived from a murine mAb and a human immunoglobulinconstant region, e.g., humanized antibodies.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423-42 (1988);Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Wardet al., Nature 334:544-54 (1989)) can be adapted to produce single chainantibodies. Single chain antibodies are formed by linking the heavy andlight chain fragments of the Fv region via an amino acid bridge,resulting in a single chain polypeptide. Techniques for the assembly offunctional Fv fragments in E. coli may also be used (Skerra et al.,Science 242:1038-1041 (1988)).

Methods of Producing Antibodies

The antibodies of the invention can be produced by any method known inthe art for the synthesis of antibodies, in particular, by chemicalsynthesis or preferably, by recombinant expression techniques.

Recombinant expression of an antibody of the invention, or fragment,derivative or analog thereof, (e.g., a heavy or light chain of anantibody of the invention or a single chain antibody of the invention),requires construction of an expression vector containing apolynucleotide that encodes the antibody. Once a polynucleotide encodingan antibody molecule or a heavy or light chain of an antibody, orportion thereof (preferably containing the heavy or light chain variabledomain), of the invention has been obtained, the vector for theproduction of the antibody molecule may be produced by recombinant DNAtechnology using techniques well known in the art. Thus, methods forpreparing a protein by expressing a polynucleotide containing anantibody encoding nucleotide sequence are described herein. Methodswhich are well known to those skilled in the art can be used toconstruct expression vectors containing antibody coding sequences andappropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. The invention,thus, provides replicable vectors comprising a nucleotide sequenceencoding an antibody molecule of the invention, or a heavy or lightchain thereof, or a heavy or light chain variable domain, operablylinked to a promoter. Such vectors may include the nucleotide sequenceencoding the constant region of the antibody molecule (see, e.g., PCTPublication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No.5,122,464) and the variable domain of the antibody may be cloned intosuch a vector for expression of the entire heavy or light chain.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an antibody of the invention. Thus, the inventionincludes host cells containing a polynucleotide encoding an antibody ofthe invention, or a heavy or light chain thereof, or a single chainantibody of the invention, operably linked to a heterologous promoter.In preferred embodiments for the expression of double-chainedantibodies, vectors encoding both the heavy and light chains may beco-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed below.

A variety of host-expression vector systems may be utilized to expressthe antibody molecules of the invention. Such host-expression systemsrepresent vehicles by which the coding sequences of interest may beproduced and subsequently purified, but also represent cells which may,when transformed or transfected with the appropriate nucleotide codingsequences, express an antibody molecule of the invention in situ. Theseinclude but are not limited to microorganisms such as bacteria (e.g., E.coli, B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing antibody codingsequences; yeast (e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing antibody codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing antibody codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing antibody coding sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinantexpression constructs containing promoters derived from the genome ofmammalian cells (e.g., metallothionein promoter) or from mammalianviruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5Kpromoter). Preferably, bacterial cells such as Escherichia coli, andmore preferably, eukaryotic cells, especially for the expression ofwhole recombinant antibody molecule, are used for the expression of arecombinant antibody molecule. For example, mammalian cells such asChinese hamster ovary cells (CHO), in conjunction with a vector such asthe major intermediate early gene promoter element from humancytomegalovirus is an effective expression system for antibodies(Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2(1990)).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791(1983)), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lac Z coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, NucleicAcids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem.24:5503-5509 (1989)); and the like. pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding tomatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The antibody coding sequence may be clonedindividually into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the antibody molecule in infected hosts. (e.g., see Logan &Shenk, Proc. Natl. Acad. Sci. USA 81:355-359 (1984)). Specificinitiation signals may also be required for efficient translation ofinserted antibody coding sequences. These signals include the ATGinitiation codon and adjacent sequences. Furthermore, the initiationcodon must be in phase with the reading frame of the desired codingsequence to ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see Bittner et al., Methodsin Enzymol. 153:51-544 (1987)).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK,293, 3T3, W138, and in particular, breast cancer cell lines such as, forexample, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary glandcell line such as, for example, CRL7030 and Hs578Bst.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe antibody molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the antibodymolecule. Such engineered cell lines may be particularly useful inscreening and evaluation of compounds that interact directly orindirectly with the antibody molecule.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223(1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adeninephosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes can beemployed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl.Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072(1981)); neo, which confers resistance to the aminoglycoside G-418Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991);Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan,Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem.62:191-217 (1993); May, 1993, TIB TECH 11(5):155-215); and hygro, whichconfers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)).Methods commonly known in the art of recombinant DNA technology may beroutinely applied to select the desired recombinant clone, and suchmethods are described, for example, in Ausubel et al. (eds.), CurrentProtocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler,Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY(1990); and in Chapters 12 and 13, Dracopoli et al. (eds), CurrentProtocols in Human Genetics, John Wiley & Sons, NY (1994);Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which areincorporated by reference herein in their entireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington and Hentschel, The use ofvectors based on gene amplification for the expression of cloned genesin mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York,1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al., Mol. Cell. Biol. 3:257(1983)).

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector may be used which encodes, and is capable of expressing,both heavy and light chain polypeptides. In such situations, the lightchain should be placed before the heavy chain to avoid an excess oftoxic free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc.Natl. Acad. Sci. USA 77:2197 (1980)). The coding sequences for the heavyand light chains may comprise cDNA or genomic DNA.

Once an antibody molecule of the invention has been produced by ananimal, chemically synthesized, or recombinantly expressed, it may bepurified by any method known in the art for purification of animmunoglobulin molecule, for example, by chromatography (e.g., ionexchange, affinity, particularly by affinity for the specific antigenafter Protein A, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of proteins. In addition, the antibodies of the presentinvention or fragments thereof can be fused to heterologous polypeptidesequences described herein or otherwise known in the art, to facilitatepurification.

The present invention encompasses antibodies recombinantly fused orchemically conjugated (including both covalently and non-covalentlyconjugations) to a polypeptide (or portion thereof, preferably at least10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of thepolypeptide) of the present invention to generate fusion proteins. Thefusion does not necessarily need to be direct, but may occur throughlinker sequences. The antibodies may be specific for antigens other thanpolypeptides (or portion thereof, preferably at least 10, 20, 30, 40,50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the presentinvention. For example, antibodies may be used to target thepolypeptides of the present invention to particular cell types, eitherin vitro or in vivo, by fusing or conjugating the polypeptides of thepresent invention to antibodies specific for particular cell surfacereceptors. Antibodies fused or conjugated to the polypeptides of thepresent invention may also be used in vitro immunoassays andpurification methods using methods known in the art. See e.g., Harbor etal., supra, and PCT publication WO 93/21232; EP 439,095; Naramura etal., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No. 5,474,981; Gillies etal., PNAS 89:1428-1432 (1992); Fell et al., J. Immunol. 146:2446-2452(1991), which are incorporated by reference in their entireties.

The present invention further includes compositions comprising thepolypeptides of the present invention fused or conjugated to antibodydomains other than the variable regions. For example, the polypeptidesof the present invention may be fused or conjugated to an antibody Fcregion, or portion thereof. The antibody portion fused to a polypeptideof the present invention may comprise the constant region, hinge region,CH1 domain, CH2 domain, and CH3 domain or any combination of wholedomains or portions thereof. The polypeptides may also be fused orconjugated to the above antibody portions to form multimers. Forexample, Fc portions fused to the polypeptides of the present inventioncan form dimers through disulfide bonding between the Fc portions.Higher multimeric forms can be made by fusing the polypeptides toportions of IgA and IgM. Methods for fusing or conjugating thepolypeptides of the present invention to antibody portions are known inthe art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046;5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166; PCTpublications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc. Natl.Acad. Sci. USA 88:10535-10539 (1991); Zheng et al., J. Immunol.154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA89:11337-11341 (1992) (said references incorporated by reference intheir entireties).

As discussed, supra, the polypeptides corresponding to a polypeptide,polypeptide fragment, or a variant of SEQ ID NO:Y may be fused orconjugated to the above antibody portions to increase the in vivo halflife of the polypeptides or for use in immunoassays using methods knownin the art. Further, the polypeptides corresponding to SEQ ID NO:Y maybe fused or conjugated to the above antibody portions to facilitatepurification. One reported example describes chimeric proteinsconsisting of the first two domains of the human CD4-polypeptide andvarious domains of the constant regions of the heavy or light chains ofmammalian immunoglobulins. (EP 394,827; Traunecker et al., Nature331:84-86 (1988). The polypeptides of the present invention fused orconjugated to an antibody having disulfide-linked dimeric structures(due to the IgG) may also be more efficient in binding and neutralizingother molecules, than the monomeric secreted protein or protein fragmentalone. (Fountoulakis et al., J. Biochem. 270:3958-3964 (1995)). In manycases, the Fc part in a fusion protein is beneficial in therapy anddiagnosis, and thus can result in, for example, improved pharmacokineticproperties. (EP A 232,262). Alternatively, deleting the Fc part afterthe fusion protein has been expressed, detected, and purified, would bedesired. For example, the Fc portion may hinder therapy and diagnosis ifthe fusion protein is used as an antigen for immunizations. In drugdiscovery, for example, human proteins, such as hIL-5, have been fusedwith Fc portions for the purpose of high-throughput screening assays toidentify antagonists of hIL-5. (See, Bennett et al., J. MolecularRecognition 8:52-58 (1995); Johanson et al., J. Biol. Chem.270:9459-9471 (1995).

Moreover, the antibodies or fragments thereof of the present inventioncan be fused to marker sequences, such as a peptide to facilitatepurification. In preferred embodiments, the marker amino acid sequenceis a hexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described in Gentzet al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance,hexa-histidine provides for convenient purification of the fusionprotein. Other peptide tags useful for purification include, but are notlimited to, the “HA” tag, which corresponds to an epitope derived fromthe influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984))and the “flag” tag.

The present invention further encompasses antibodies or fragmentsthereof conjugated to a diagnostic or therapeutic agent. The antibodiescan be used diagnostically to, for example, monitor the development orprogression of a tumor as part of a clinical testing procedure to, e.g.,determine the efficacy of a given treatment regimen. Detection can befacilitated by coupling the antibody to a detectable substance. Examplesof detectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,radioactive materials, positron emitting metals using various positronemission tomographies, and nonradioactive paramagnetic metal ions. Thedetectable substance may be coupled or conjugated either directly to theantibody (or fragment thereof) or indirectly, through an intermediate(such as, for example, a linker known in the art) using techniques knownin the art. See, for example, U.S. Pat. No. 4,741,900 for metal ionswhich can be conjugated to antibodies for use as diagnostics accordingto the present invention. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude 125I, 131I, 111In or 99Tc.

Further, an antibody or fragment thereof may be conjugated to atherapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidalagent, a therapeutic agent or a radioactive metal ion, e.g.,alpha-emitters such as, for example, 213Bi. A cytotoxin or cytotoxicagent includes any agent that is detrimental to cells. Examples includepaclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologues thereof. Therapeutic agents include, but are not limitedto, antimetabolites (e.g., methotrexate, 6-mercaptopurine,6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylatingagents (e.g., mechlorethamine, thioepa chlorambucil, melphalan,carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan,dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin(formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin(formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)),and anti-mitotic agents (e.g., vincristine and vinblastine).

The conjugates of the invention can be used for modifying a givenbiological response, the therapeutic agent or drug moiety is not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis factor, -interferon, β-interferon,nerve growth factor, platelet derived growth factor, tissue plasminogenactivator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I (See,International Publication No. WO 97/33899), AIM II (See, InternationalPublication No. WO 97/34911), Fas Ligand (Takahashi et al., Int.Immunol., 6:1567-1574 (1994)), VEGI (See, International Publication No.WO 99/23105), a thrombotic agent or an anti-angiogenic agent, e.g.,angiostatin or endostatin; or, biological response modifiers such as,for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev. 62:119-58 (1982).

Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described by Segal in U.S. Pat. No.4,676,980, which is incorporated herein by reference in its entirety.

An antibody, with or without a therapeutic moiety conjugated to it,administered alone or in combination with cytotoxic factor(s) and/orcytokine(s) can be used as a therapeutic.

Uses for Antibodies Directed Against Polypeptides of the Invention

The antibodies of the present invention have various utilities. Forexample, such antibodies may be used in diagnostic assays to detect thepresence or quantification of the polypeptides of the invention in asample. Such a diagnostic assay may be comprised of at least two steps.The first, subjecting a sample with the antibody, wherein the sample isa tissue (e.g., human, animal, etc.), biological fluid (e.g., blood,urine, sputum, semen, amniotic fluid, saliva, etc.), biological extract(e.g., tissue or cellular homogenate, etc.), a protein microchip (e.g.,See Arenkov P, et al., Anal Biochem., 278(2):123-131 (2000)), or achromatography column, etc. And a second step involving thequantification of antibody bound to the substrate. Alternatively, themethod may additionally involve a first step of attaching the antibody,either covalently, electrostatically, or reversibly, to a solid support,and a second step of subjecting the bound antibody to the sample, asdefined above and elsewhere herein.

Various diagnostic assay techniques are known in the art, such ascompetitive binding assays, direct or indirect sandwich assays andimmunoprecipitation assays conducted in either heterogeneous orhomogenous phases (Zola, Monoclonal Antibodies: A Manual of Techniques,CRC Press, Inc., (1987), pp 147-158). The antibodies used in thediagnostic assays can be labeled with a detectable moiety. Thedetectable moiety should be capable of producing, either directly orindirectly, a detectable signal. For example, the detectable moiety maybe a radioisotope, such as 2H, 14C, 32P, or 125I, a florescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase,beta-galactosidase, green fluorescent protein, or horseradishperoxidase. Any method known in the art for conjugating the antibody tothe detectable moiety may be employed, including those methods describedby Hunter et al., Nature, 144:945 (1962); Dafvid et al., Biochem.,13:1014 (1974); Pain et al., J. Immunol. Metho., 40:219 (1981); andNygren, J. Histochem. And Cytochem., 30:407 (1982).

Antibodies directed against the polypeptides of the present inventionare useful for the affinity purification of such polypeptides fromrecombinant cell culture or natural sources. In this process, theantibodies against a particular polypeptide are immobilized on asuitable support, such as a Sephadex resin or filter paper, usingmethods well known in the art. The immobilized antibody then iscontacted with a sample containing the polypeptides to be purified, andthereafter the support is washed with a suitable solvent that willremove substantially all the material in the sample except for thedesired polypeptides, which are bound to the immobilized antibody.Finally, the support is washed with another suitable solvent that willrelease the desired polypeptide from the antibody.

Immunophenotyping

The antibodies of the invention may be utilized for immunophenotyping ofcell lines and biological samples. The translation product of the geneof the present invention may be useful as a cell specific marker, ormore specifically as a cellular marker that is differentially expressedat various stages of differentiation and/or maturation of particularcell types. Monoclonal antibodies directed against a specific epitope,or combination of epitopes, will allow for the screening of cellularpopulations expressing the marker. Various techniques can be utilizedusing monoclonal antibodies to screen for cellular populationsexpressing the marker(s), and include magnetic separation usingantibody-coated magnetic beads, “panning” with antibody attached to asolid matrix (i.e., plate), and flow cytometry (See, e.g., U.S. Pat. No.5,985,660; and Morrison et al., Cell, 96:737-49 (1999)).

These techniques allow for the screening of particular populations ofcells, such as might be found with hematological malignancies (i.e.minimal residual disease (MRD) in acute leukemic patients) and“non-self” cells in transplantations to prevent Graft-versus-HostDisease (GVHD). Alternatively, these techniques allow for the screeningof hematopoietic stem and progenitor cells capable of undergoingproliferation and/or differentiation, as might be found in humanumbilical cord blood.

Assays for Antibody Binding

The antibodies of the invention may be assayed for immunospecificbinding by any method known in the art. The immunoassays which can beused include but are not limited to competitive and non-competitiveassay systems using techniques such as western blots, radioimmunoassays,ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, to name but a few. Such assays areroutine and well known in the art (see, e.g., Ausubel et al, eds, 1994,Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc.,New York, which is incorporated by reference herein in its entirety).Exemplary immunoassays are described briefly below (but are not intendedby way of limitation).

Immunoprecipitation protocols generally comprise lysing a population ofcells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100,1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphateat pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/orprotease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate),adding the antibody of interest to the cell lysate, incubating for aperiod of time (e.g., 1-4 hours) at 4° C., adding protein A and/orprotein G sepharose beads to the cell lysate, incubating for about anhour or more at 4° C., washing the beads in lysis buffer andresuspending the beads in SDS/sample buffer. The ability of the antibodyof interest to immunoprecipitate a particular antigen can be assessedby, e.g., western blot analysis. One of skill in the art would beknowledgeable as to the parameters that can be modified to increase thebinding of the antibody to an antigen and decrease the background (e.g.,pre-clearing the cell lysate with sepharose beads). For furtherdiscussion regarding immunoprecipitation protocols see, e.g., Ausubel etal, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York at 10.16.1.

Western blot analysis generally comprises preparing protein samples,electrophoresis of the protein samples in a polyacrylamide gel (e.g.,8%-20% SDS-PAGE depending on the molecular weight of the antigen),transferring the protein sample from the polyacrylamide gel to amembrane such as nitrocellulose, PVDF or nylon, blocking the membrane inblocking solution (e.g., PBS with 3% BSA or non-fat milk), washing themembrane in washing buffer (e.g., PBS-Tween 20), blocking the membranewith primary antibody (the antibody of interest) diluted in blockingbuffer, washing the membrane in washing buffer, blocking the membranewith a secondary antibody (which recognizes the primary antibody, e.g.,an anti-human antibody) conjugated to an enzymatic substrate (e.g.,horseradish peroxidase or alkaline phosphatase) or radioactive molecule(e.g., 32P or 125I) diluted in blocking buffer, washing the membrane inwash buffer, and detecting the presence of the antigen. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected and to reduce the background noise. Forfurther discussion regarding western blot protocols see, e.g., Ausubelet al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York at 10.8.1.

ELISAs comprise preparing antigen, coating the well of a 96 wellmicrotiter plate with the antigen, adding the antibody of interestconjugated to a detectable compound such as an enzymatic substrate(e.g., horseradish peroxidase or alkaline phosphatase) to the well andincubating for a period of time, and detecting the presence of theantigen. In ELISAs the antibody of interest does not have to beconjugated to a detectable compound; instead, a second antibody (whichrecognizes the antibody of interest) conjugated to a detectable compoundmay be added to the well. Further, instead of coating the well with theantigen, the antibody may be coated to the well. In this case, a secondantibody conjugated to a detectable compound may be added following theaddition of the antigen of interest to the coated well. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected as well as other variations of ELISAsknown in the art. For further discussion regarding ELISAs see, e.g.,Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol.1, John Wiley & Sons, Inc., New York at 11.2.1.

The binding affinity of an antibody to an antigen and the off-rate of anantibody-antigen interaction can be determined by competitive bindingassays. One example of a competitive binding assay is a radioimmunoassaycomprising the incubation of labeled antigen (e.g., 3H or 125I) with theantibody of interest in the presence of increasing amounts of unlabeledantigen, and the detection of the antibody bound to the labeled antigen.The affinity of the antibody of interest for a particular antigen andthe binding off-rates can be determined from the data by scatchard plotanalysis. Competition with a second antibody can also be determinedusing radioimmunoassays. In this case, the antigen is incubated withantibody of interest conjugated to a labeled compound (e.g., 3H or 125I)in the presence of increasing amounts of an unlabeled second antibody.

Therapeutic Uses of Antibodies

The present invention is further directed to antibody-based therapieswhich involve administering antibodies of the invention to an animal,preferably a mammal, and most preferably a human, patient for treatingone or more of the disclosed diseases, disorders, or conditions.Therapeutic compounds of the invention include, but are not limited to,antibodies of the invention (including fragments, analogs andderivatives thereof as described herein) and nucleic acids encodingantibodies of the invention (including fragments, analogs andderivatives thereof and anti-idiotypic antibodies as described herein).The antibodies of the invention can be used to treat, inhibit or preventdiseases, disorders or conditions associated with aberrant expressionand/or activity of a polypeptide of the invention, including, but notlimited to, any one or more of the diseases, disorders, or conditionsdescribed herein. The treatment and/or prevention of diseases,disorders, or conditions associated with aberrant expression and/oractivity of a polypeptide of the invention includes, but is not limitedto, alleviating symptoms associated with those diseases, disorders orconditions. Antibodies of the invention may be provided inpharmaceutically acceptable compositions as known in the art or asdescribed herein.

A summary of the ways in which the antibodies of the present inventionmay be used therapeutically includes binding polynucleotides orpolypeptides of the present invention locally or systemically in thebody or by direct cytotoxicity of the antibody, e.g. as mediated bycomplement (CDC) or by effector cells (ADCC). Some of these approachesare described in more detail below. Armed with the teachings providedherein, one of ordinary skill in the art will know how to use theantibodies of the present invention for diagnostic, monitoring ortherapeutic purposes without undue experimentation.

The antibodies of this invention may be advantageously utilized incombination with other monoclonal or chimeric antibodies, or withlymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3and IL-7), for example, which serve to increase the number or activityof effector cells which interact with the antibodies.

The antibodies of the invention may be administered alone or incombination with other types of treatments (e.g., radiation therapy,chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents).Generally, administration of products of a species origin or speciesreactivity (in the case of antibodies) that is the same species as thatof the patient is preferred. Thus, in a preferred embodiment, humanantibodies, fragments derivatives, analogs, or nucleic acids, areadministered to a human patient for therapy or prophylaxis.

It is preferred to use high affinity and/or potent in vivo inhibitingand/or neutralizing antibodies against polypeptides or polynucleotidesof the present invention, fragments or regions thereof, for bothimmunoassays directed to and therapy of disorders related topolynucleotides or polypeptides, including fragments thereof, of thepresent invention. Such antibodies, fragments, or regions, willpreferably have an affinity for polynucleotides or polypeptides of theinvention, including fragments thereof. Preferred binding affinitiesinclude those with a dissociation constant or Kd less than 5×10-2 M,10-2M, 5×10-3 M, 10-3 M, 5×10-4 M, 10-4 M, 5×10-5 M, 10-5 M, 5×10-6 M,10-6 M, 5×10-7 M, 10-7 M, 5×10-8 M, 10-8 M, 5×10-9 M, 10-9 M, 5×10-10 M,10-10 M, 5×10-11 M, 10-11 M, 5×10-12 M, 10-12 M, 5×10-13 M, 10-13 M,5×10-14 M, 10-14 M, 5×10-15 M, and 10-15 M.

Antibodies directed against polypeptides of the present invention areuseful for inhibiting allergic reactions in animals. For example, byadministering a therapeutically acceptable dose of an antibody, orantibodies, of the present invention, or a cocktail of the presentantibodies, or in combination with other antibodies of varying sources,the animal may not elicit an allergic response to antigens.

Likewise, one could envision cloning the gene encoding an antibodydirected against a polypeptide of the present invention, saidpolypeptide having the potential to elicit an allergic and/or immuneresponse in an organism, and transforming the organism with saidantibody gene such that it is expressed (e.g., constitutively,inducibly, etc.) in the organism. Thus, the organism would effectivelybecome resistant to an allergic response resulting from the ingestion orpresence of such an immune/allergic reactive polypeptide. Moreover, sucha use of the antibodies of the present invention may have particularutility in preventing and/or ameliorating autoimmune diseases and/ordisorders, as such conditions are typically a result of antibodies beingdirected against endogenous proteins. For example, in the instance wherethe polypeptide of the present invention is responsible for modulatingthe immune response to auto-antigens, transforming the organism and/orindividual with a construct comprising any of the promoters disclosedherein or otherwise known in the art, in addition, to a polynucleotideencoding the antibody directed against the polypeptide of the presentinvention could effective inhibit the organisms immune system fromeliciting an immune response to the auto-antigen(s). Detaileddescriptions of therapeutic and/or gene therapy applications of thepresent invention are provided elsewhere herein.

Alternatively, antibodies of the present invention could be produced ina plant (e.g., cloning the gene of the antibody directed against apolypeptide of the present invention, and transforming a plant with asuitable vector comprising said gene for constitutive expression of theantibody within the plant), and the plant subsequently ingested by ananimal, thereby conferring temporary immunity to the animal for thespecific antigen the antibody is directed towards (See, for example,U.S. Pat. Nos. 5,914,123 and 6,034,298).

In another embodiment, antibodies of the present invention, preferablypolyclonal antibodies, more preferably monoclonal antibodies, and mostpreferably single-chain antibodies, can be used as a means of inhibitinggene expression of a particular gene, or genes, in a human, mammal,and/or other organism. See, for example, International PublicationNumber WO 00/05391, published Feb. 3, 2000, to Dow Agrosciences LLC. Theapplication of such methods for the antibodies of the present inventionare known in the art, and are more particularly described elsewhereherein.

In yet another embodiment, antibodies of the present invention may beuseful for multimerizing the polypeptides of the present invention. Forexample, certain proteins may confer enhanced biological activity whenpresent in a multimeric state (i.e., such enhanced activity may be dueto the increased effective concentration of such proteins whereby moreprotein is available in a localized location).

Antibody-Based Gene Therapy

In a specific embodiment, nucleic acids comprising sequences encodingantibodies or functional derivatives thereof, are administered to treat,inhibit or prevent a disease or disorder associated with aberrantexpression and/or activity of a polypeptide of the invention, by way ofgene therapy. Gene therapy refers to therapy performed by theadministration to a subject of an expressed or expressible nucleic acid.In this embodiment of the invention, the nucleic acids produce theirencoded protein that mediates a therapeutic effect.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. Exemplary methods are describedbelow.

For general reviews of the methods of gene therapy, see Goldspiel etal., Clinical Pharmacy 12:488-505 (1993); Wu and Wu, Biotherapy 3:87-95(1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993);Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev.Biochem. 62:191-217 (1993); May, TIBTECH 11(5):155-215 (1993). Methodscommonly known in the art of recombinant DNA technology which can beused are described in Ausubel et al. (eds.), Current Protocols inMolecular Biology, John Wiley & Sons, NY (1993); and Kriegler, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

In a preferred aspect, the compound comprises nucleic acid sequencesencoding an antibody, said nucleic acid sequences being part ofexpression vectors that express the antibody or fragments or chimericproteins or heavy or light chains thereof in a suitable host. Inparticular, such nucleic acid sequences have promoters operably linkedto the antibody coding region, said promoter being inducible orconstitutive, and, optionally, tissue-specific. In another particularembodiment, nucleic acid molecules are used in which the antibody codingsequences and any other desired sequences are flanked by regions thatpromote homologous recombination at a desired site in the genome, thusproviding for intrachromosomal expression of the antibody encodingnucleic acids (Koller and Smithies, Proc. Natl. Acad. Sci. USA86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989). Inspecific embodiments, the expressed antibody molecule is a single chainantibody; alternatively, the nucleic acid sequences include sequencesencoding both the heavy and light chains, or fragments thereof, of theantibody.

Delivery of the nucleic acids into a patient may be either direct, inwhich case the patient is directly exposed to the nucleic acid ornucleic acid-carrying vectors, or indirect, in which case, cells arefirst transformed with the nucleic acids in vitro, then transplantedinto the patient. These two approaches are known, respectively, as invivo or ex vivo gene therapy.

In a specific embodiment, the nucleic acid sequences are directlyadministered in vivo, where it is expressed to produce the encodedproduct. This can be accomplished by any of numerous methods known inthe art, e.g., by constructing them as part of an appropriate nucleicacid expression vector and administering it so that they becomeintracellular, e.g., by infection using defective or attenuatedretrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or bydirect injection of naked DNA, or by use of microparticle bombardment(e.g., a gene gun; Biolistic, Dupont), or coating with lipids orcell-surface receptors or transfecting agents, encapsulation inliposomes, microparticles, or microcapsules, or by administering them inlinkage to a peptide which is known to enter the nucleus, byadministering it in linkage to a ligand subject to receptor-mediatedendocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987))(which can be used to target cell types specifically expressing thereceptors), etc. In another embodiment, nucleic acid-ligand complexescan be formed in which the ligand comprises a fusogenic viral peptide todisrupt endosomes, allowing the nucleic acid to avoid lysosomaldegradation. In yet another embodiment, the nucleic acid can be targetedin vivo for cell specific uptake and expression, by targeting a specificreceptor (see, e.g., PCT Publications WO 92/06180; WO 92/22635;WO92/20316; WO93/14188, WO 93/20221). Alternatively, the nucleic acidcan be introduced intracellularly and incorporated within host cell DNAfor expression, by homologous recombination (Koller and Smithies, Proc.Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature342:435-438 (1989)).

In a specific embodiment, viral vectors that contains nucleic acidsequences encoding an antibody of the invention are used. For example, aretroviral vector can be used (see Miller et al., Meth. Enzymol.217:581-599 (1993)). These retroviral vectors contain the componentsnecessary for the correct packaging of the viral genome and integrationinto the host cell DNA. The nucleic acid sequences encoding the antibodyto be used in gene therapy are cloned into one or more vectors, whichfacilitates delivery of the gene into a patient. More detail aboutretroviral vectors can be found in Boesen et al., Biotherapy 6:291-302(1994), which describes the use of a retroviral vector to deliver themdr1 gene to hematopoietic stem cells in order to make the stem cellsmore resistant to chemotherapy. Other references illustrating the use ofretroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest.93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons andGunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson,Curr. Opin. in Genetics and Devel. 3:110-114 (1993).

Adenoviruses are other viral vectors that can be used in gene therapy.Adenoviruses are especially attractive vehicles for delivering genes torespiratory epithelia. Adenoviruses naturally infect respiratoryepithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, CurrentOpinion in Genetics and Development 3:499-503 (1993) present a review ofadenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10(1994) demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al.,Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992);Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT PublicationWO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). In apreferred embodiment, adenovirus vectors are used.

Adeno-associated virus (AAV) has also been proposed for use in genetherapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993);U.S. Pat. No. 5,436,146).

Another approach to gene therapy involves transferring a gene to cellsin tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a patient.

In this embodiment, the nucleic acid is introduced into a cell prior toadministration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign genes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol.217:599-618 (1993); Cohen et al., Meth. Enzymol. 217:618-644 (1993);Cline, Pharmac. Ther. 29:69-92m (1985) and may be used in accordancewith the present invention, provided that the necessary developmentaland physiological functions of the recipient cells are not disrupted.The technique should provide for the stable transfer of the nucleic acidto the cell, so that the nucleic acid is expressible by the cell andpreferably heritable and expressible by its cell progeny.

The resulting recombinant cells can be delivered to a patient by variousmethods known in the art. Recombinant blood cells (e.g., hematopoieticstem or progenitor cells) are preferably administered intravenously. Theamount of cells envisioned for use depends on the desired effect,patient state, etc., and can be determined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and include but arenot limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such asTlymphocytes, Blymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

In a preferred embodiment, the cell used for gene therapy is autologousto the patient.

In an embodiment in which recombinant cells are used in gene therapy,nucleic acid sequences encoding an antibody are introduced into thecells such that they are expressible by the cells or their progeny, andthe recombinant cells are then administered in vivo for therapeuticeffect. In a specific embodiment, stem or progenitor cells are used. Anystem and/or progenitor cells which can be isolated and maintained invitro can potentially be used in accordance with this embodiment of thepresent invention (see e.g. PCT Publication WO 94/08598; Stemple andAnderson, Cell 71:973-985 (1992); Rheinwald, Meth. Cell Bio. 21A:229(1980); and Pittelkow and Scott, Mayo Clinic Proc. 61:771 (1986)).

In a specific embodiment, the nucleic acid to be introduced for purposesof gene therapy comprises an inducible promoter operably linked to thecoding region, such that expression of the nucleic acid is controllableby controlling the presence or absence of the appropriate inducer oftranscription. Demonstration of Therapeutic or Prophylactic Activity

The compounds or pharmaceutical compositions of the invention arepreferably tested in vitro, and then in vivo for the desired therapeuticor prophylactic activity, prior to use in humans. For example, in vitroassays to demonstrate the therapeutic or prophylactic utility of acompound or pharmaceutical composition include, the effect of a compoundon a cell line or a patient tissue sample. The effect of the compound orcomposition on the cell line and/or tissue sample can be determinedutilizing techniques known to those of skill in the art including, butnot limited to, rosette formation assays and cell lysis assays. Inaccordance with the invention, in vitro assays which can be used todetermine whether administration of a specific compound is indicated,include in vitro cell culture assays in which a patient tissue sample isgrown in culture, and exposed to or otherwise administered a compound,and the effect of such compound upon the tissue sample is observed.

Therapeutic/Prophylactic Administration and Compositions

The invention provides methods of treatment, inhibition and prophylaxisby administration to a subject of an effective amount of a compound orpharmaceutical composition of the invention, preferably an antibody ofthe invention. In a preferred aspect, the compound is substantiallypurified (e.g., substantially free from substances that limit its effector produce undesired side-effects). The subject is preferably an animal,including but not limited to animals such as cows, pigs, horses,chickens, cats, dogs, etc., and is preferably a mammal, and mostpreferably human.

Formulations and methods of administration that can be employed when thecompound comprises a nucleic acid or an immunoglobulin are describedabove; additional appropriate formulations and routes of administrationcan be selected from among those described herein below.

Various delivery systems are known and can be used to administer acompound of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J.Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid aspart of a retroviral or other vector, etc. Methods of introductioninclude but are not limited to intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The compounds or compositions may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. Administration can besystemic or local. In addition, it may be desirable to introduce thepharmaceutical compounds or compositions of the invention into thecentral nervous system by any suitable route, including intraventricularand intrathecal injection; intraventricular injection may be facilitatedby an intraventricular catheter, for example, attached to a reservoir,such as an Ommaya reservoir. Pulmonary administration can also beemployed, e.g., by use of an inhaler or nebulizer, and formulation withan aerosolizing agent.

In a specific embodiment, it may be desirable to administer thepharmaceutical compounds or compositions of the invention locally to thearea in need of treatment; this may be achieved by, for example, and notby way of limitation, local infusion during surgery, topicalapplication, e.g., in conjunction with a wound dressing after surgery,by injection, by means of a catheter, by means of a suppository, or bymeans of an implant, said implant being of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers. Preferably, when administering a protein, including anantibody, of the invention, care must be taken to use materials to whichthe protein does not absorb.

In another embodiment, the compound or composition can be delivered in avesicle, in particular a liposome (see Langer, Science 249:1527-1533(1990); Treat et al., in Liposomes in the Therapy of Infectious Diseaseand Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp.353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generallyibid.)

In yet another embodiment, the compound or composition can be deliveredin a controlled release system. In one embodiment, a pump may be used(see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987);Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med.321:574 (1989)). In another embodiment, polymeric materials can be used(see Medical Applications of Controlled Release, Langer and Wise (eds.),CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability,Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, NewYork (1984); Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem.23:61 (1983); see also Levy et al., Science 228:190 (1985); During etal., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105(1989)). In yet another embodiment, a controlled release system can beplaced in proximity of the therapeutic target, i.e., the brain, thusrequiring only a fraction of the systemic dose (see, e.g., Goodson, inMedical Applications of Controlled Release, supra, vol. 2, pp. 115-138(1984)).

Other controlled release systems are discussed in the review by Langer(Science 249:1527-1533 (1990)).

In a specific embodiment where the compound of the invention is anucleic acid encoding a protein, the nucleic acid can be administered invivo to promote expression of its encoded protein, by constructing it aspart of an appropriate nucleic acid expression vector and administeringit so that it becomes intracellular, e.g., by use of a retroviral vector(see U.S. Pat. No. 4,980,286), or by direct injection, or by use ofmicroparticle bombardment (e.g., a gene gun; Biolistic, Dupont), orcoating with lipids or cell-surface receptors or transfecting agents, orby administering it in linkage to a homeobox-like peptide which is knownto enter the nucleus (see e.g., Joliot et al., Proc. Natl. Acad. Sci.USA 88:1864-1868 (1991)), etc. Alternatively, a nucleic acid can beintroduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination.

The present invention also provides pharmaceutical compositions. Suchcompositions comprise a therapeutically effective amount of a compound,and a pharmaceutically acceptable carrier. In a specific embodiment, theterm “pharmaceutically acceptable” means approved by a regulatory agencyof the Federal or a state government or listed in the U.S. Pharmacopeiaor other generally recognized pharmacopeia for use in animals, and moreparticularly in humans. The term “carrier” refers to a diluent,adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. Such compositions will containa therapeutically effective amount of the compound, preferably inpurified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the patient. Theformulation should suit the mode of administration.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The compounds of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed withanions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

The amount of the compound of the invention which will be effective inthe treatment, inhibition and prevention of a disease or disorderassociated with aberrant expression and/or activity of a polypeptide ofthe invention can be determined by standard clinical techniques. Inaddition, in vitro assays may optionally be employed to help identifyoptimal dosage ranges. The precise dose to be employed in theformulation will also depend on the route of administration, and theseriousness of the disease or disorder, and should be decided accordingto the judgment of the practitioner and each patient's circumstances.Effective doses may be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems.

For antibodies, the dosage administered to a patient is typically 0.1mg/kg to 100 mg/kg of the patient's body weight. Preferably, the dosageadministered to a patient is between 0.1 mg/kg and 20 mg/kg of thepatient's body weight, more preferably 1 mg/kg to 10 mg/kg of thepatient's body weight. Generally, human antibodies have a longerhalf-life within the human body than antibodies from other species dueto the immune response to the foreign polypeptides. Thus, lower dosagesof human antibodies and less frequent administration is often possible.Further, the dosage and frequency of administration of antibodies of theinvention may be reduced by enhancing uptake and tissue penetration(e.g., into the brain) of the antibodies by modifications such as, forexample, lipidation.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

Diagnosis and Imaging with Antibodies

Labeled antibodies, and derivatives and analogs thereof, whichspecifically bind to a polypeptide of interest can be used fordiagnostic purposes to detect, diagnose, or monitor diseases, disorders,and/or conditions associated with the aberrant expression and/oractivity of a polypeptide of the invention. The invention provides forthe detection of aberrant expression of a polypeptide of interest,comprising (a) assaying the expression of the polypeptide of interest incells or body fluid of an individual using one or more antibodiesspecific to the polypeptide interest and (b) comparing the level of geneexpression with a standard gene expression level, whereby an increase ordecrease in the assayed polypeptide gene expression level compared tothe standard expression level is indicative of aberrant expression.

The invention provides a diagnostic assay for diagnosing a disorder,comprising (a) assaying the expression of the polypeptide of interest incells or body fluid of an individual using one or more antibodiesspecific to the polypeptide interest and (b) comparing the level of geneexpression with a standard gene expression level, whereby an increase ordecrease in the assayed polypeptide gene expression level compared tothe standard expression level is indicative of a particular disorder.With respect to cancer, the presence of a relatively high amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Antibodies of the invention can be used to assay protein levels in abiological sample using classical immunohistological methods known tothose of skill in the art (e.g., see Jalkanen, et al., J. Cell. Biol.101:976-985 (1985); Jalkanen, et al., J. Cell. Biol. 105:3087-3096(1987)). Other antibody-based methods useful for detecting protein geneexpression include immunoassays, such as the enzyme linked immunosorbentassay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assaylabels are known in the art and include enzyme labels, such as, glucoseoxidase; radioisotopes, such as iodine (125I, 121I), carbon (14C),sulfur (35S), tritium (3H), indium (112In), and technetium (99Tc);luminescent labels, such as luminol; and fluorescent labels, such asfluorescein and rhodamine, and biotin.

One aspect of the invention is the detection and diagnosis of a diseaseor disorder associated with aberrant expression of a polypeptide ofinterest in an animal, preferably a mammal and most preferably a human.In one embodiment, diagnosis comprises: a) administering (for example,parenterally, subcutaneously, or intraperitoneally) to a subject aneffective amount of a labeled molecule which specifically binds to thepolypeptide of interest; b) waiting for a time interval following theadministering for permitting the labeled molecule to preferentiallyconcentrate at sites in the subject where the polypeptide is expressed(and for unbound labeled molecule to be cleared to background level); c)determining background level; and d) detecting the labeled molecule inthe subject, such that detection of labeled molecule above thebackground level indicates that the subject has a particular disease ordisorder associated with aberrant expression of the polypeptide ofinterest. Background level can be determined by various methodsincluding, comparing the amount of labeled molecule detected to astandard value previously determined for a particular system.

It will be understood in the art that the size of the subject and theimaging system used will determine the quantity of imaging moiety neededto produce diagnostic images. In the case of a radioisotope moiety, fora human subject, the quantity of radioactivity injected will normallyrange from about 5 to 20 millicuries of 99mTc. The labeled antibody orantibody fragment will then preferentially accumulate at the location ofcells which contain the specific protein. In vivo tumor imaging isdescribed in S. W. Burchiel et al., “Immunopharmacokinetics ofRadiolabeled Antibodies and Their Fragments.” (Chapter 13 in TumorImaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A.Rhodes, eds., Masson Publishing Inc. (1982).

Depending on several variables, including the type of label used and themode of administration, the time interval following the administrationfor permitting the labeled molecule to preferentially concentrate atsites in the subject and for unbound labeled molecule to be cleared tobackground level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. Inanother embodiment the time interval following administration is 5 to 20days or 5 to 10 days.

In an embodiment, monitoring of the disease or disorder is carried outby repeating the method for diagnosing the disease or disease, forexample, one month after initial diagnosis, six months after initialdiagnosis, one year after initial diagnosis, etc.

Presence of the labeled molecule can be detected in the patient usingmethods known in the art for in vivo scanning. These methods depend uponthe type of label used. Skilled artisans will be able to determine theappropriate method for detecting a particular label. Methods and devicesthat may be used in the diagnostic methods of the invention include, butare not limited to, computed tomography (CT), whole body scan such asposition emission tomography (PET), magnetic resonance imaging (MRI),and sonography.

In a specific embodiment, the molecule is labeled with a radioisotopeand is detected in the patient using a radiation responsive surgicalinstrument (Thurston et al., U.S. Pat. No. 5,441,050). In anotherembodiment, the molecule is labeled with a fluorescent compound and isdetected in the patient using a fluorescence responsive scanninginstrument. In another embodiment, the molecule is labeled with apositron emitting metal and is detected in the patent using positronemission-tomography. In yet another embodiment, the molecule is labeledwith a paramagnetic label and is detected in a patient using magneticresonance imaging (MRI).

Kits

The present invention provides kits that can be used in the abovemethods. In one embodiment, a kit comprises an antibody of theinvention, preferably a purified antibody, in one or more containers. Ina specific embodiment, the kits of the present invention contain asubstantially isolated polypeptide comprising an epitope which isspecifically immunoreactive with an antibody included in the kit.Preferably, the kits of the present invention further comprise a controlantibody which does not react with the polypeptide of interest. Inanother specific embodiment, the kits of the present invention contain ameans for detecting the binding of an antibody to a polypeptide ofinterest (e.g., the antibody may be conjugated to a detectable substratesuch as a fluorescent compound, an enzymatic substrate, a radioactivecompound or a luminescent compound, or a second antibody whichrecognizes the first antibody may be conjugated to a detectablesubstrate).

In another specific embodiment of the present invention, the kit is adiagnostic kit for use in screening serum containing antibodies specificagainst proliferative and/or cancerous polynucleotides and polypeptides.Such a kit may include a control antibody that does not react with thepolypeptide of interest. Such a kit may include a substantially isolatedpolypeptide antigen comprising an epitope which is specificallyimmunoreactive with at least one anti-polypeptide antigen antibody.Further, such a kit includes means for detecting the binding of saidantibody to the antigen (e.g., the antibody may be conjugated to afluorescent compound such as fluorescein or rhodamine which can bedetected by flow cytometry). In specific embodiments, the kit mayinclude a recombinantly produced or chemically synthesized polypeptideantigen. The polypeptide antigen of the kit may also be attached to asolid support.

In a more specific embodiment the detecting means of the above-describedkit includes a solid support to which said polypeptide antigen isattached. Such a kit may also include a non-attached reporter-labeledanti-human antibody. In this embodiment, binding of the antibody to thepolypeptide antigen can be detected by binding of the saidreporter-labeled antibody.

In an additional embodiment, the invention includes a diagnostic kit foruse in screening serum containing antigens of the polypeptide of theinvention. The diagnostic kit includes a substantially isolated antibodyspecifically immunoreactive with polypeptide or polynucleotide antigens,and means for detecting the binding of the polynucleotide or polypeptideantigen to the antibody. In one embodiment, the antibody is attached toa solid support. In a specific embodiment, the antibody may be amonoclonal antibody. The detecting means of the kit may include asecond, labeled monoclonal antibody. Alternatively, or in addition, thedetecting means may include a labeled, competing antigen.

In one diagnostic configuration, test serum is reacted with a solidphase reagent having a surface-bound antigen obtained by the methods ofthe present invention. After binding with specific antigen antibody tothe reagent and removing unbound serum components by washing, thereagent is reacted with reporter-labeled anti-human antibody to bindreporter to the reagent in proportion to the amount of boundanti-antigen antibody on the solid support. The reagent is again washedto remove unbound labeled antibody, and the amount of reporterassociated with the reagent is determined. Typically, the reporter is anenzyme which is detected by incubating the solid phase in the presenceof a suitable fluorometric, luminescent or colorimetric substrate(Sigma, St. Louis, Mo.).

The solid surface reagent in the above assay is prepared by knowntechniques for attaching protein material to solid support material,such as polymeric beads, dip sticks, 96-well plate or filter material.These attachment methods generally include non-specific adsorption ofthe protein to the support or covalent attachment of the protein,typically through a free amine group, to a chemically reactive group onthe solid support, such as an activated carboxyl, hydroxyl, or aldehydegroup. Alternatively, streptavidin coated plates can be used inconjunction with biotinylated antigen(s).

Thus, the invention provides an assay system or kit for carrying outthis diagnostic method. The kit generally includes a support withsurface-bound recombinant antigens, and a reporter-labeled anti-humanantibody for detecting surface-bound anti-antigen antibody.

Fusion Proteins

Any polypeptide of the present invention can be used to generate fusionproteins. For example, the polypeptide of the present invention, whenfused to a second protein, can be used as an antigenic tag. Antibodiesraised against the polypeptide of the present invention can be used toindirectly detect the second protein by binding to the polypeptide.Moreover, because certain proteins target cellular locations based ontrafficking signals, the polypeptides of the present invention can beused as targeting molecules once fused to other proteins.

Examples of domains that can be fused to polypeptides of the presentinvention include not only heterologous signal sequences, but also otherheterologous functional regions. The fusion does not necessarily need tobe direct, but may occur through linker sequences.

Moreover, fusion proteins may also be engineered to improvecharacteristics of the polypeptide of the present invention. Forinstance, a region of additional amino acids, particularly charged aminoacids, may be added to the N-terminus of the polypeptide to improvestability and persistence during purification from the host cell orsubsequent handling and storage. Peptide moieties may be added to thepolypeptide to facilitate purification. Such regions may be removedprior to final preparation of the polypeptide. Similarly, peptidecleavage sites can be introduced in-between such peptide moieties, whichcould additionally be subjected to protease activity to remove saidpeptide(s) from the protein of the present invention. The addition ofpeptide moieties, including peptide cleavage sites, to facilitatehandling of polypeptides are familiar and routine techniques in the art.

Moreover, polypeptides of the present invention, including fragments,and specifically epitopes, can be combined with parts of the constantdomain of immunoglobulins (IgA, IgE, IgG, IgM) or portions thereof (CH1,CH2, CH3, and any combination thereof, including both entire domains andportions thereof), resulting in chimeric polypeptides. These fusionproteins facilitate purification and show an increased half-life invivo. One reported example describes chimeric proteins consisting of thefirst two domains of the human CD4-polypeptide and various domains ofthe constant regions of the heavy or light chains of mammalianimmunoglobulins. (EP A 394,827; Traunecker et al., Nature 331:84-86(1988).) Fusion proteins having disulfide-linked dimeric structures (dueto the IgG) can also be more efficient in binding and neutralizing othermolecules, than the monomeric secreted protein or protein fragmentalone. (Fountoulakis et al., J. Biochem. 270:3958-3964 (1995).)

Similarly, EP-A-O 464 533 (Canadian counterpart 2045869) disclosesfusion proteins comprising various portions of the constant region ofimmunoglobulin molecules together with another human protein or partthereof. In many cases, the Fc part in a fusion protein is beneficial intherapy and diagnosis, and thus can result in, for example, improvedpharmacokinetic properties. (EP-A 0232 262.) Alternatively, deleting theFc part after the fusion protein has been expressed, detected, andpurified, would be desired. For example, the Fc portion may hindertherapy and diagnosis if the fusion protein is used as an antigen forimmunizations. In drug discovery, for example, human proteins, such ashIL-5, have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. (See,D. Bennett et al., J. Molecular Recognition 8:52-58 (1995); K. Johansonet al., J. Biol. Chem. 270:9459-9471 (1995).)

Moreover, the polypeptides of the present invention can be fused tomarker sequences (also referred to as “tags”). Due to the availabilityof antibodies specific to such “tags”, purification of the fusedpolypeptide of the invention, and/or its identification is significantlyfacilitated since antibodies specific to the polypeptides of theinvention are not required. Such purification may be in the form of anaffinity purification whereby an anti-tag antibody or another type ofaffinity matrix (e.g., anti-tag antibody attached to the matrix of aflow-thru column) that binds to the epitope tag is present. In preferredembodiments, the marker amino acid sequence is a hexa-histidine peptide,such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 EtonAvenue, Chatsworth, Calif., 91311), among others, many of which arecommercially available. As described in Gentz et al., Proc. Natl. Acad.Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides forconvenient purification of the fusion protein. Another peptide taguseful for purification, the “HA” tag, corresponds to an epitope derivedfrom the influenza hemagglutinin protein. (Wilson et al., Cell 37:767(1984)).

The skilled artisan would acknowledge the existence of other “tags”which could be readily substituted for the tags referred to supra forpurification and/or identification of polypeptides of the presentinvention (Jones C., et al., J Chromatogr A. 707(1):3-22 (1995)). Forexample, the c-myc tag and the 8F9, 3C7, 6E10, G4m B7 and 9E10antibodies thereto (Evan et al., Molecular and Cellular Biology5:3610-3616 (1985)); the Herpes Simplex virus glycoprotein D (gD) tagand its antibody (Paborsky et al., Protein Engineering, 3(6):547-553(1990), the Flag-peptide—i.e., the octapeptide sequence DYKDDDDK (SEQ IDNO:75), (Hopp et al., Biotech. 6:1204-1210 (1988); the KT3 epitopepeptide (Martin et al., Science, 255:192-194 (1992)); a-tubulin epitopepeptide (Skinner et al., J. Biol. Chem., 266:15136-15166, (1991)); theT7 gene 10 protein peptide tag (Lutz-Freyermuth et al., Proc. Natl. Sci.USA, 87:6363-6397 (1990)), the FITC epitope (Zymed, Inc.), the GFPepitope (Zymed, Inc.), and the Rhodamine epitope (Zymed, Inc.).

The present invention also encompasses the attachment of up to ninecodons encoding a repeating series of up to nine arginine amino acids tothe coding region of a polynucleotide of the present invention. Theinvention also encompasses chemically derivitizing a polypeptide of thepresent invention with a repeating series of up to nine arginine aminoacids. Such a tag, when attached to a polypeptide, has recently beenshown to serve as a universal pass, allowing compounds access to theinterior of cells without additional derivitization or manipulation(Wender, P., et al., unpublished data).

Protein fusions involving polypeptides of the present invention,including fragments and/or variants thereof, can be used for thefollowing, non-limiting examples, subcellular localization of proteins,determination of protein-protein interactions via immunoprecipitation,purification of proteins via affinity chromatography, functional and/orstructural characterization of protein. The present invention alsoencompasses the application of hapten specific antibodies for any of theuses referenced above for epitope fusion proteins. For example, thepolypeptides of the present invention could be chemically derivatized toattach hapten molecules (e.g., DNP, (Zymed, Inc.)). Due to theavailability of monoclonal antibodies specific to such haptens, theprotein could be readily purified using immunoprecipation, for example.

Polypeptides of the present invention, including fragments and/orvariants thereof, in addition to, antibodies directed against suchpolypeptides, fragments, and/or variants, may be fused to any of anumber of known, and yet to be determined, toxins, such as ricin,saporin (Mashiba H, et al., Ann. N.Y. Acad. Sci. 1999; 886:233-5), or HCtoxin (Tonukari N J, et al., Plant Cell. 2000 February; 12(2):237-248),for example. Such fusions could be used to deliver the toxins to desiredtissues for which a ligand or a protein capable of binding to thepolypeptides of the invention exists.

The invention encompasses the fusion of antibodies directed againstpolypeptides of the present invention, including variants and fragmentsthereof, to said toxins for delivering the toxin to specific locationsin a cell, to specific tissues, and/or to specific species. Suchbifunctional antibodies are known in the art, though a review describingadditional advantageous fusions, including citations for methods ofproduction, can be found in P. J. Hudson, Curr. Opp. In. Imm.11:548-557, (1999); this publication, in addition to the referencescited therein, are hereby incorporated by reference in their entiretyherein. In this context, the term “toxin” may be expanded to include anyheterologous protein, a small molecule, radionucleotides, cytotoxicdrugs, liposomes, adhesion molecules, glycoproteins, ligands, cell ortissue-specific ligands, enzymes, of bioactive agents, biologicalresponse modifiers, anti-fungal agents, hormones, steroids, vitamins,peptides, peptide analogs, anti-allergenic agents, anti-tubercularagents, anti-viral agents, antibiotics, anti-protozoan agents, chelates,radioactive particles, radioactive ions, X-ray contrast agents,monoclonal antibodies, polyclonal antibodies and genetic material. Inview of the present disclosure, one skilled in the art could determinewhether any particular “toxin” could be used in the compounds of thepresent invention. Examples of suitable “toxins” listed above areexemplary only and are not intended to limit the “toxins” that may beused in the present invention.

Thus, any of these above fusions can be engineered using thepolynucleotides or the polypeptides of the present invention.

Vectors, Host Cells, and Protein Production

The present invention also relates to vectors containing thepolynucleotide of the present invention, host cells, and the productionof polypeptides by recombinant techniques. The vector may be, forexample, a phage, plasmid, viral, or retroviral vector. Retroviralvectors may be replication competent or replication defective. In thelatter case, viral propagation generally will occur only incomplementing host cells.

The polynucleotides may be joined to a vector containing a selectablemarker for propagation in a host. Generally, a plasmid vector isintroduced in a precipitate, such as a calcium phosphate precipitate, orin a complex with a charged lipid. If the vector is a virus, it may bepackaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

The polynucleotide insert should be operatively linked to an appropriatepromoter, such as the phage lambda PL promoter, the E. coli lac, trp,phoA and tac promoters, the SV40 early and late promoters and promotersof retroviral LTRs, to name a few. Other suitable promoters will beknown to the skilled artisan. The expression constructs will furthercontain sites for transcription initiation, termination, and, in thetranscribed region, a ribosome binding site for translation. The codingportion of the transcripts expressed by the constructs will preferablyinclude a translation initiating codon at the beginning and atermination codon (UAA, UGA or UAG) appropriately positioned at the endof the polypeptide to be translated.

As indicated, the expression vectors will preferably include at leastone selectable marker. Such markers include dihydrofolate reductase,G418 or neomycin resistance for eukaryotic cell culture andtetracycline, kanamycin or ampicillin resistance genes for culturing inE. coli and other bacteria. Representative examples of appropriate hostsinclude, but are not limited to, bacterial cells, such as E. coli,Streptomyces and Salmonella typhimurium cells; fungal cells, such asyeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCCAccession No. 201178)); insect cells such as Drosophila S2 andSpodoptera Sf9 cells; animal cells such as CHO, COS, 293, and Bowesmelanoma cells; and plant cells. Appropriate culture mediums andconditions for the above-described host cells are known in the art.

Among vectors preferred for use in bacteria include pQE70, pQE60 andpQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescriptvectors, pNH8A, pNH16a, pNH18A, pNH46A, available from StratageneCloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5available from Pharmacia Biotech, Inc. Among preferred eukaryoticvectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available fromStratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.Preferred expression vectors for use in yeast systems include, but arenot limited to pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ,pGAPZalph, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K, andPAO815 (all available from Invitrogen, Carlsbad, Calif.). Other suitablevectors will be readily apparent to the skilled artisan.

Introduction of the construct into the host cell can be effected bycalcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection, or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., Basic Methods In MolecularBiology (1986). It is specifically contemplated that the polypeptides ofthe present invention may in fact be expressed by a host cell lacking arecombinant vector.

A polypeptide of this invention can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Most preferably, highperformance liquid chromatography (“HPLC”) is employed for purification.

Polypeptides of the present invention, and preferably the secreted form,can also be recovered from: products purified from natural sources,including bodily fluids, tissues and cells, whether directly isolated orcultured; products of chemical synthetic procedures; and productsproduced by recombinant techniques from a prokaryotic or eukaryotichost, including, for example, bacterial, yeast, higher plant, insect,and mammalian cells. Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. In addition, polypeptides ofthe invention may also include an initial modified methionine residue,in some cases as a result of host-mediated processes. Thus, it is wellknown in the art that the N-terminal methionine encoded by thetranslation initiation codon generally is removed with high efficiencyfrom any protein after translation in all eukaryotic cells. While theN-terminal methionine on most proteins also is efficiently removed inmost prokaryotes, for some proteins, this prokaryotic removal process isinefficient, depending on the nature of the amino acid to which theN-terminal methionine is covalently linked.

In one embodiment, the yeast Pichia pastoris is used to express thepolypeptide of the present invention in a eukaryotic system. Pichiapastoris is a methylotrophic yeast which can metabolize methanol as itssole carbon source. A main step in the methanol metabolization pathwayis the oxidation of methanol to formaldehyde using O2. This reaction iscatalyzed by the enzyme alcohol oxidase. In order to metabolize methanolas its sole carbon source, Pichia pastoris must generate high levels ofalcohol oxidase due, in part, to the relatively low affinity of alcoholoxidase for O2. Consequently, in a growth medium depending on methanolas a main carbon source, the promoter region of one of the two alcoholoxidase genes (AOX1) is highly active. In the presence of methanol,alcohol oxidase produced from the AOX1 gene comprises up toapproximately 30% of the total soluble protein in Pichia pastoris. See,Ellis, S. B., et al., Mol. Cell. Biol. 5:1111-21 (1985); Koutz, P. J, etal., Yeast 5:167-77 (1989); Tschopp, J. F., et al., Nucl. Acids Res.15:3859-76 (1987). Thus, a heterologous coding sequence, such as, forexample, a polynucleotide of the present invention, under thetranscriptional regulation of all or part of the AOX1 regulatorysequence is expressed at exceptionally high levels in Pichia yeast grownin the presence of methanol.

In one example, the plasmid vector pPIC9K is used to express DNAencoding a polypeptide of the invention, as set forth herein, in aPichea yeast system essentially as described in “Pichia Protocols:Methods in Molecular Biology,” D. R. Higgins and J. Cregg, eds. TheHumana Press, Totowa, N.J., 1998. This expression vector allowsexpression and secretion of a protein of the invention by virtue of thestrong AOX1 promoter linked to the Pichia pastoris alkaline phosphatase(PHO) secretory signal peptide (i.e., leader) located upstream of amultiple cloning site.

Many other yeast vectors could be used in place of pPIC9K, such as,pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9,pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, and PAO815, as one skilled in theart would readily appreciate, as long as the proposed expressionconstruct provides appropriately located signals for transcription,translation, secretion (if desired), and the like, including an in-frameAUG, as required.

In another embodiment, high-level expression of a heterologous codingsequence, such as, for example, a polynucleotide of the presentinvention, may be achieved by cloning the heterologous polynucleotide ofthe invention into an expression vector such as, for example, pGAPZ orpGAPZalpha, and growing the yeast culture in the absence of methanol.

In addition to encompassing host cells containing the vector constructsdiscussed herein, the invention also encompasses primary, secondary, andimmortalized host cells of vertebrate origin, particularly mammalianorigin, that have been engineered to delete or replace endogenousgenetic material (e.g., coding sequence), and/or to include geneticmaterial (e.g., heterologous polynucleotide sequences) that is operablyassociated with the polynucleotides of the invention, and whichactivates, alters, and/or amplifies endogenous polynucleotides. Forexample, techniques known in the art may be used to operably associateheterologous control regions (e.g., promoter and/or enhancer) andendogenous polynucleotide sequences via homologous recombination,resulting in the formation of a new transcription unit (see, e.g., U.S.Pat. No. 5,641,670, issued Jun. 24, 1997; U.S. Pat. No. 5,733,761,issued Mar. 31, 1998; International Publication No. WO 96/29411,published Sep. 26, 1996; International Publication No. WO 94/12650,published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989), thedisclosures of each of which are incorporated by reference in theirentireties).

In addition, polypeptides of the invention can be chemically synthesizedusing techniques known in the art (e.g., see Creighton, 1983, Proteins:Structures and Molecular Principles, W.H. Freeman & Co., N.Y., andHunkapiller et al., Nature, 310:105-111 (1984)). For example, apolypeptide corresponding to a fragment of a polypeptide sequence of theinvention can be synthesized by use of a peptide synthesizer.Furthermore, if desired, nonclassical amino acids or chemical amino acidanalogs can be introduced as a substitution or addition into thepolypeptide sequence. Non-classical amino acids include, but are notlimited to, to the D-isomers of the common amino acids,2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid,Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib,2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine,norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline,cysteic acid, t-butylglycine, t-butylalanine, phenylglycine,cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acidssuch as b-methyl amino acids, Ca-methyl amino acids, Na-methyl aminoacids, and amino acid analogs in general. Furthermore, the amino acidcan be D (dextrorotary) or L (levorotary).

The invention encompasses polypeptides which are differentially modifiedduring or after translation, e.g., by glycosylation, acetylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to an antibody molecule or othercellular ligand, etc. Any of numerous chemical modifications may becarried out by known techniques, including but not limited, to specificchemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8protease, NaBH4; acetylation, formylation, oxidation, reduction;metabolic synthesis in the presence of tunicamycin; etc.

Additional post-translational modifications encompassed by the inventioninclude, for example, e.g., N-linked or O-linked carbohydrate chains,processing of N-terminal or C-terminal ends), attachment of chemicalmoieties to the amino acid backbone, chemical modifications of N-linkedor O-linked carbohydrate chains, and addition or deletion of anN-terminal methionine residue as a result of prokaryotic host cellexpression. The polypeptides may also be modified with a detectablelabel, such as an enzymatic, fluorescent, isotopic or affinity label toallow for detection and isolation of the protein, the addition ofepitope tagged peptide fragments (e.g., FLAG, HA, GST, thioredoxin,maltose binding protein, etc.), attachment of affinity tags such asbiotin and/or streptavidin, the covalent attachment of chemical moietiesto the amino acid backbone, N- or C-terminal processing of thepolypeptides ends (e.g., proteolytic processing), deletion of theN-terminal methionine residue, etc.

Also provided by the invention are chemically modified derivatives ofthe polypeptides of the invention which may provide additionaladvantages such as increased solubility, stability and circulating timeof the polypeptide, or decreased immunogenicity (see U.S. Pat. No.4,179,337). The chemical moieties for derivitization may be selectedfrom water soluble polymers such as polyethylene glycol, ethyleneglycol/propylene glycol copolymers, carboxymethylcellulose, dextran,polyvinyl alcohol and the like. The polypeptides may be modified atrandom positions within the molecule, or at predetermined positionswithin the molecule and may include one, two, three or more attachedchemical moieties.

The invention further encompasses chemical derivitization of thepolypeptides of the present invention, preferably where the chemical isa hydrophilic polymer residue. Exemplary hydrophilic polymers, includingderivatives, may be those that include polymers in which the repeatingunits contain one or more hydroxy groups (polyhydroxy polymers),including, for example, poly(vinyl alcohol); polymers in which therepeating units contain one or more amino groups (polyamine polymers),including, for example, peptides, polypeptides, proteins andlipoproteins, such as albumin and natural lipoproteins; polymers inwhich the repeating units contain one or more carboxy groups(polycarboxy polymers), including, for example, carboxymethylcellulose,alginic acid and salts thereof, such as sodium and calcium alginate,glycosaminoglycans and salts thereof, including salts of hyaluronicacid, phosphorylated and sulfonated derivatives of carbohydrates,genetic material, such as interleukin-2 and interferon, andphosphorothioate oligomers; and polymers in which the repeating unitscontain one or more saccharide moieties (polysaccharide polymers),including, for example, carbohydrates.

The molecular weight of the hydrophilic polymers may vary, and isgenerally about 50 to about 5,000,000, with polymers having a molecularweight of about 100 to about 50,000 being preferred. The polymers may bebranched or unbranched. More preferred polymers have a molecular weightof about 150 to about 10,000, with molecular weights of 200 to about8,000 being even more preferred.

For polyethylene glycol, the preferred molecular weight is between about1 kDa and about 100 kDa (the term “about” indicating that inpreparations of polyethylene glycol, some molecules will weigh more,some less, than the stated molecular weight) for ease in handling andmanufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog).

Additional preferred polymers which may be used to derivatizepolypeptides of the invention, include, for example, poly(ethyleneglycol) (PEG), poly(vinylpyrrolidine), polyoxomers, polysorbate andpoly(vinyl alcohol), with PEG polymers being particularly preferred.Preferred among the PEG polymers are PEG polymers having a molecularweight of from about 100 to about 10,000. More preferably, the PEGpolymers have a molecular weight of from about 200 to about 8,000, withPEG 2,000, PEG 5,000 and PEG 8,000, which have molecular weights of2,000, 5,000 and 8,000, respectively, being even more preferred. Othersuitable hydrophilic polymers, in addition to those exemplified above,will be readily apparent to one skilled in the art based on the presentdisclosure. Generally, the polymers used may include polymers that canbe attached to the polypeptides of the invention via alkylation oracylation reactions.

The polyethylene glycol molecules (or other chemical moieties) should beattached to the protein with consideration of effects on functional orantigenic domains of the protein. There are a number of attachmentmethods available to those skilled in the art, e.g., EP 0 401 384,herein incorporated by reference (coupling PEG to G-CSF), see also Maliket al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation ofGM-CSF using tresyl chloride). For example, polyethylene glycol may becovalently bound through amino acid residues via a reactive group, suchas, a free amino or carboxyl group. Reactive groups are those to whichan activated polyethylene glycol molecule may be bound. The amino acidresidues having a free amino group may include lysine residues and theN-terminal amino acid residues; those having a free carboxyl group mayinclude aspartic acid residues glutamic acid residues and the C-terminalamino acid residue. Sulfhydryl groups may also be used as a reactivegroup for attaching the polyethylene glycol molecules. Preferred fortherapeutic purposes is attachment at an amino group, such as attachmentat the N-terminus or lysine group.

One may specifically desire proteins chemically modified at theN-terminus. Using polyethylene glycol as an illustration of the presentcomposition, one may select from a variety of polyethylene glycolmolecules (by molecular weight, branching, etc.), the proportion ofpolyethylene glycol molecules to protein (polypeptide) molecules in thereaction mix, the type of pegylation reaction to be performed, and themethod of obtaining the selected N-terminally pegylated protein. Themethod of obtaining the N-terminally pegylated preparation (i.e.,separating this moiety from other monopegylated moieties if necessary)may be by purification of the N-terminally pegylated material from apopulation of pegylated protein molecules. Selective proteins chemicallymodified at the N-terminus modification may be accomplished by reductivealkylation which exploits differential reactivity of different types ofprimary amino groups (lysine versus the N-terminus) available forderivatization in a particular protein. Under the appropriate reactionconditions, substantially selective derivatization of the protein at theN-terminus with a carbonyl group containing polymer is achieved.

As with the various polymers exemplified above, it is contemplated thatthe polymeric residues may contain functional groups in addition, forexample, to those typically involved in linking the polymeric residuesto the polypeptides of the present invention. Such functionalitiesinclude, for example, carboxyl, amine, hydroxy and thiol groups. Thesefunctional groups on the polymeric residues can be further reacted, ifdesired, with materials that are generally reactive with such functionalgroups and which can assist in targeting specific tissues in the bodyincluding, for example, diseased tissue. Exemplary materials which canbe reacted with the additional functional groups include, for example,proteins, including antibodies, carbohydrates, peptides, glycopeptides,glycolipids, lectins, and nucleosides.

In addition to residues of hydrophilic polymers, the chemical used toderivatize the polypeptides of the present invention can be a saccharideresidue. Exemplary saccharides which can be derived include, forexample, monosaccharides or sugar alcohols, such as erythrose, threose,ribose, arabinose, xylose, lyxose, fructose, sorbitol, mannitol andsedoheptulose, with preferred monosaccharides being fructose, mannose,xylose, arabinose, mannitol and sorbitol; and disaccharides, such aslactose, sucrose, maltose and cellobiose. Other saccharides include, forexample, inositol and ganglioside head groups. Other suitablesaccharides, in addition to those exemplified above, will be readilyapparent to one skilled in the art based on the present disclosure.Generally, saccharides which may be used for derivitization includesaccharides that can be attached to the polypeptides of the inventionvia alkylation or acylation reactions.

Moreover, the invention also encompasses derivitization of thepolypeptides of the present invention, for example, with lipids(including cationic, anionic, polymerized, charged, synthetic,saturated, unsaturated, and any combination of the above, etc.).stabilizing agents.

The invention encompasses derivitization of the polypeptides of thepresent invention, for example, with compounds that may serve astabilizing function (e.g., to increase the polypeptides half-life insolution, to make the polypeptides more water soluble, to increase thepolypeptides hydrophilic or hydrophobic character, etc.). Polymersuseful as stabilizing materials may be of natural, semi-synthetic(modified natural) or synthetic origin. Exemplary natural polymersinclude naturally occurring polysaccharides, such as, for example,arabinans, fructans, fucans, galactans, galacturonans, glucans, mannans,xylans (such as, for example, inulin), levan, fucoidan, carrageenan,galatocarolose, pectic acid, pectins, including amylose, pullulan,glycogen, amylopectin, cellulose, dextran, dextrin, dextrose, glucose,polyglucose, polydextrose, pustulan, chitin, agarose, keratin,chondroitin, dermatan, hyaluronic acid, alginic acid, xanthin gum,starch and various other natural homopolymer or heteropolymers, such asthose containing one or more of the following aldoses, ketoses, acids oramines: erythose, threose, ribose, arabinose, xylose, lyxose, allose,altrose, glucose, dextrose, mannose, gulose, idose, galactose, talose,erythrulose, ribulose, xylulose, psicose, fructose, sorbose, tagatose,mannitol, sorbitol, lactose, sucrose, trehalose, maltose, cellobiose,glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine,aspartic acid, glutamic acid, lysine, arginine, histidine, glucuronicacid, gluconic acid, glucaric acid, galacturonic acid, mannuronic acid,glucosamine, galactosamine, and neuraminic acid, and naturally occurringderivatives thereof. Accordingly, suitable polymers include, forexample, proteins, such as albumin, polyalginates, andpolylactide-coglycolide polymers. Exemplary semi-synthetic polymersinclude carboxymethylcellulose, hydroxymethylcellulose,hydroxypropylmethylcellulose, methylcellulose, and methoxycellulose.Exemplary synthetic polymers include polyphosphazenes, hydroxyapatites,fluoroapatite polymers, polyethylenes (such as, for example,polyethylene glycol (including for example, the class of compoundsreferred to as Pluronics.RTM., commercially available from BASF,Parsippany, N.J.), polyoxyethylene, and polyethylene terephthlate),polypropylenes (such as, for example, polypropylene glycol),polyurethanes (such as, for example, polyvinyl alcohol (PVA), polyvinylchloride and polyvinylpyrrolidone), polyamides including nylon,polystyrene, polylactic acids, fluorinated hydrocarbon polymers,fluorinated carbon polymers (such as, for example,polytetrafluoroethylene), acrylate, methacrylate, andpolymethylmethacrylate, and derivatives thereof. Methods for thepreparation of derivatized polypeptides of the invention which employpolymers as stabilizing compounds will be readily apparent to oneskilled in the art, in view of the present disclosure, when coupled withinformation known in the art, such as that described and referred to inUnger, U.S. Pat. No. 5,205,290, the disclosure of which is herebyincorporated by reference herein in its entirety.

Moreover, the invention encompasses additional modifications of thepolypeptides of the present invention. Such additional modifications areknown in the art, and are specifically provided, in addition to methodsof derivitization, etc., in U.S. Pat. No. 6,028,066, which is herebyincorporated in its entirety herein.

The polypeptides of the invention may be in monomers or multimers (i.e.,dimers, trimers, tetramers and higher multimers). Accordingly, thepresent invention relates to monomers and multimers of the polypeptidesof the invention, their preparation, and compositions (preferably,Therapeutics) containing them. In specific embodiments, the polypeptidesof the invention are monomers, dimers, trimers or tetramers. Inadditional embodiments, the multimers of the invention are at leastdimers, at least trimers, or at least tetramers.

Multimers encompassed by the invention may be homomers or heteromers. Asused herein, the term homomer, refers to a multimer containing onlypolypeptides corresponding to the amino acid sequence of SEQ ID NO:Y orencoded by the cDNA contained in a deposited clone (including fragments,variants, splice variants, and fusion proteins, corresponding to thesepolypeptides as described herein). These homomers may containpolypeptides having identical or different amino acid sequences. In aspecific embodiment, a homomer of the invention is a multimer containingonly polypeptides having an identical amino acid sequence. In anotherspecific embodiment, a homomer of the invention is a multimer containingpolypeptides having different amino acid sequences. In specificembodiments, the multimer of the invention is a homodimer (e.g.,containing polypeptides having identical or different amino acidsequences) or a homotrimer (e.g., containing polypeptides havingidentical and/or different amino acid sequences). In additionalembodiments, the homomeric multimer of the invention is at least ahomodimer, at least a homotrimer, or at least a homotetramer.

As used herein, the term heteromer refers to a multimer containing oneor more heterologous polypeptides (i.e., polypeptides of differentproteins) in addition to the polypeptides of the invention. In aspecific embodiment, the multimer of the invention is a heterodimer, aheterotrimer, or a heterotetramer. In additional embodiments, theheteromeric multimer of the invention is at least a heterodimer, atleast a heterotrimer, or at least a heterotetramer.

Multimers of the invention may be the result of hydrophobic,hydrophilic, ionic and/or covalent associations and/or may be indirectlylinked, by for example, liposome formation. Thus, in one embodiment,multimers of the invention, such as, for example, homodimers orhomotrimers, are formed when polypeptides of the invention contact oneanother in solution. In another embodiment, heteromultimers of theinvention, such as, for example, heterotrimers or heterotetramers, areformed when polypeptides of the invention contact antibodies to thepolypeptides of the invention (including antibodies to the heterologouspolypeptide sequence in a fusion protein of the invention) in solution.In other embodiments, multimers of the invention are formed by covalentassociations with and/or between the polypeptides of the invention. Suchcovalent associations may involve one or more amino acid residuescontained in the polypeptide sequence (e.g., that recited in thesequence listing, or contained in the polypeptide encoded by a depositedclone). In one instance, the covalent associations are cross-linkingbetween cysteine residues located within the polypeptide sequences whichinteract in the native (i.e., naturally occurring) polypeptide. Inanother instance, the covalent associations are the consequence ofchemical or recombinant manipulation. Alternatively, such covalentassociations may involve one or more amino acid residues contained inthe heterologous polypeptide sequence in a fusion protein of theinvention.

In one example, covalent associations are between the heterologoussequence contained in a fusion protein of the invention (see, e.g., U.S.Pat. No. 5,478,925). In a specific example, the covalent associationsare between the heterologous sequence contained in an Fc fusion proteinof the invention (as described herein). In another specific example,covalent associations of fusion proteins of the invention are betweenheterologous polypeptide sequence from another protein that is capableof forming covalently associated multimers, such as for example,osteoprotegerin (see, e.g., International Publication NO: WO 98/49305,the contents of which are herein incorporated by reference in itsentirety). In another embodiment, two or more polypeptides of theinvention are joined through peptide linkers. Examples include thosepeptide linkers described in U.S. Pat. No. 5,073,627 (herebyincorporated by reference). Proteins comprising multiple polypeptides ofthe invention separated by peptide linkers may be produced usingconventional recombinant DNA technology.

Another method for preparing multimer polypeptides of the inventioninvolves use of polypeptides of the invention fused to a leucine zipperor isoleucine zipper polypeptide sequence. Leucine zipper and isoleucinezipper domains are polypeptides that promote multimerization of theproteins in which they are found. Leucine zippers were originallyidentified in several DNA-binding proteins (Landschulz et al., Science240:1759, (1988)), and have since been found in a variety of differentproteins. Among the known leucine zippers are naturally occurringpeptides and derivatives thereof that dimerize or trimerize. Examples ofleucine zipper domains suitable for producing soluble multimericproteins of the invention are those described in PCT application WO94/10308, hereby incorporated by reference. Recombinant fusion proteinscomprising a polypeptide of the invention fused to a polypeptidesequence that dimerizes or trimerizes in solution are expressed insuitable host cells, and the resulting soluble multimeric fusion proteinis recovered from the culture supernatant using techniques known in theart.

Trimeric polypeptides of the invention may offer the advantage ofenhanced biological activity. Preferred leucine zipper moieties andisoleucine moieties are those that preferentially form trimers. Oneexample is a leucine zipper derived from lung surfactant protein D(SPD), as described in Hoppe et al. (FEBS Letters 344:191, (1994)) andin U.S. patent application Ser. No. 08/446,922, hereby incorporated byreference. Other peptides derived from naturally occurring trimericproteins may be employed in preparing trimeric polypeptides of theinvention.

In another example, proteins of the invention are associated byinteractions between Flag® polypeptide sequence contained in fusionproteins of the invention containing Flag® polypeptide sequence. In afurther embodiment, associations proteins of the invention areassociated by interactions between heterologous polypeptide sequencecontained in Flag® fusion proteins of the invention and anti-Flag®antibody.

The multimers of the invention may be generated using chemicaltechniques known in the art. For example, polypeptides desired to becontained in the multimers of the invention may be chemicallycross-linked using linker molecules and linker molecule lengthoptimization techniques known in the art (see, e.g., U.S. Pat. No.5,478,925, which is herein incorporated by reference in its entirety).Additionally, multimers of the invention may be generated usingtechniques known in the art to form one or more inter-moleculecross-links between the cysteine residues located within the sequence ofthe polypeptides desired to be contained in the multimer (see, e.g.,U.S. Pat. No. 5,478,925, which is herein incorporated by reference inits entirety). Further, polypeptides of the invention may be routinelymodified by the addition of cysteine or biotin to the C terminus orN-terminus of the polypeptide and techniques known in the art may beapplied to generate multimers containing one or more of these modifiedpolypeptides (see, e.g., U.S. Pat. No. 5,478,925, which is hereinincorporated by reference in its entirety). Additionally, techniquesknown in the art may be applied to generate liposomes containing thepolypeptide components desired to be contained in the multimer of theinvention (see, e.g., U.S. Pat. No. 5,478,925, which is hereinincorporated by reference in its entirety).

Alternatively, multimers of the invention may be generated using geneticengineering techniques known in the art. In one embodiment, polypeptidescontained in multimers of the invention are produced recombinantly usingfusion protein technology described herein or otherwise known in the art(see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated byreference in its entirety). In a specific embodiment, polynucleotidescoding for a homodimer of the invention are generated by ligating apolynucleotide sequence encoding a polypeptide of the invention to asequence encoding a linker polypeptide and then further to a syntheticpolynucleotide encoding the translated product of the polypeptide in thereverse orientation from the original C-terminus to the N-terminus(lacking the leader sequence) (see, e.g., U.S. Pat. No. 5,478,925, whichis herein incorporated by reference in its entirety). In anotherembodiment, recombinant techniques described herein or otherwise knownin the art are applied to generate recombinant polypeptides of theinvention which contain a transmembrane domain (or hydrophobic or signalpeptide) and which can be incorporated by membrane reconstitutiontechniques into liposomes (see, e.g., U.S. Pat. No. 5,478,925, which isherein incorporated by reference in its entirety).

In addition, the polynucleotide insert of the present invention could beoperatively linked to “artificial” or chimeric promoters andtranscription factors. Specifically, the artificial promoter couldcomprise, or alternatively consist, of any combination of cis-acting DNAsequence elements that are recognized by trans-acting transcriptionfactors. Preferably, the cis acting DNA sequence elements andtrans-acting transcription factors are operable in mammals. Further, thetrans-acting transcription factors of such “artificial” promoters couldalso be “artificial” or chimeric in design themselves and could act asactivators or repressors to said “artificial” promoter.

Uses of the Polynucleotides

Each of the polynucleotides identified herein can be used in numerousways as reagents. The following description should be consideredexemplary and utilizes known techniques.

The polynucleotides of the present invention are useful for chromosomeidentification. There exists an ongoing need to identify new chromosomemarkers, since few chromosome marking reagents, based on actual sequencedata (repeat polymorphisms), are presently available. Eachpolynucleotide of the present invention can be used as a chromosomemarker.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers(preferably 15-25 bp) from the sequences shown in SEQ ID NO:X. Primerscan be selected using computer analysis so that primers do not span morethan one predicted exon in the genomic DNA. These primers are then usedfor PCR screening of somatic cell hybrids containing individual humanchromosomes. Only those hybrids containing the human gene correspondingto the SEQ ID NO:X will yield an amplified fragment.

Similarly, somatic hybrids provide a rapid method of PCR mapping thepolynucleotides to particular chromosomes. Three or more clones can beassigned per day using a single thermal cycler. Moreover,sublocalization of the polynucleotides can be achieved with panels ofspecific chromosome fragments. Other gene mapping strategies that can beused include in situ hybridization, prescreening with labeledflow-sorted chromosomes, and preselection by hybridization to constructchromosome specific-cDNA libraries.

Precise chromosomal location of the polynucleotides can also be achievedusing fluorescence in situ hybridization (FISH) of a metaphasechromosomal spread. This technique uses polynucleotides as short as 500or 600 bases; however, polynucleotides 2,000-4,000 bp are preferred. Fora review of this technique, see Verma et al., “Human Chromosomes: aManual of Basic Techniques,” Pergamon Press, New York (1988).

For chromosome mapping, the polynucleotides can be used individually (tomark a single chromosome or a single site on that chromosome) or inpanels (for marking multiple sites and/or multiple chromosomes).Preferred polynucleotides correspond to the noncoding regions of thecDNAs because the coding sequences are more likely conserved within genefamilies, thus increasing the chance of cross hybridization duringchromosomal mapping.

Once a polynucleotide has been mapped to a precise chromosomal location,the physical position of the polynucleotide can be used in linkageanalysis. Linkage analysis establishes coinheritance between achromosomal location and presentation of a particular disease. Diseasemapping data are known in the art. Assuming 1 megabase mappingresolution and one gene per 20 kb, a cDNA precisely localized to achromosomal region associated with the disease could be one of 50-500potential causative genes.

Thus, once coinheritance is established, differences in thepolynucleotide and the corresponding gene between affected andunaffected organisms can be examined. First, visible structuralalterations in the chromosomes, such as deletions or translocations, areexamined in chromosome spreads or by PCR. If no structural alterationsexist, the presence of point mutations are ascertained. Mutationsobserved in some or all affected organisms, but not in normal organisms,indicates that the mutation may cause the disease. However, completesequencing of the polypeptide and the corresponding gene from severalnormal organisms is required to distinguish the mutation from apolymorphism. If a new polymorphism is identified, this polymorphicpolypeptide can be used for further linkage analysis.

Furthermore, increased or decreased expression of the gene in affectedorganisms as compared to unaffected organisms can be assessed usingpolynucleotides of the present invention. Any of these alterations(altered expression, chromosomal rearrangement, or mutation) can be usedas a diagnostic or prognostic marker.

Thus, the invention also provides a diagnostic method useful duringdiagnosis of a disorder, involving measuring the expression level ofpolynucleotides of the present invention in cells or body fluid from anorganism and comparing the measured gene expression level with astandard level of polynucleotide expression level, whereby an increaseor decrease in the gene expression level compared to the standard isindicative of a disorder.

By “measuring the expression level of a polynucleotide of the presentinvention” is intended qualitatively or quantitatively measuring orestimating the level of the polypeptide of the present invention or thelevel of the mRNA encoding the polypeptide in a first biological sampleeither directly (e.g., by determining or estimating absolute proteinlevel or mRNA level) or relatively (e.g., by comparing to thepolypeptide level or mRNA level in a second biological sample).Preferably, the polypeptide level or mRNA level in the first biologicalsample is measured or estimated and compared to a standard polypeptidelevel or mRNA level, the standard being taken from a second biologicalsample obtained from an individual not having the disorder or beingdetermined by averaging levels from a population of organisms not havinga disorder. As will be appreciated in the art, once a standardpolypeptide level or mRNA level is known, it can be used repeatedly as astandard for comparison.

By “biological sample” is intended any biological sample obtained froman organism, body fluids, cell line, tissue culture, or other sourcewhich contains the polypeptide of the present invention or mRNA. Asindicated, biological samples include body fluids (such as the followingnon-limiting examples, sputum, amniotic fluid, urine, saliva, breastmilk, secretions, interstitial fluid, blood, serum, spinal fluid, etc.)which contain the polypeptide of the present invention, and other tissuesources found to express the polypeptide of the present invention.Methods for obtaining tissue biopsies and body fluids from organisms arewell known in the art. Where the biological sample is to include mRNA, atissue biopsy is the preferred source.

The method(s) provided above may Preferably be applied in a diagnosticmethod and/or kits in which polynucleotides and/or polypeptides areattached to a solid support. In one exemplary method, the support may bea “gene chip” or a “biological chip” as described in U.S. Pat. Nos.5,837,832, 5,874,219, and 5,856,174. Further, such a gene chip withpolynucleotides of the present invention attached may be used toidentify polymorphisms between the polynucleotide sequences, withpolynucleotides isolated from a test subject. The knowledge of suchpolymorphisms (i.e. their location, as well as, their existence) wouldbe beneficial in identifying disease loci for many disorders, includingproliferative diseases and conditions. Such a method is described inU.S. Pat. Nos. 5,858,659 and 5,856,104. The US patents referenced supraare hereby incorporated by reference in their entirety herein.

The present invention encompasses polynucleotides of the presentinvention that are chemically synthesized, or reproduced as peptidenucleic acids (PNA), or according to other methods known in the art. Theuse of PNAs would serve as the preferred form if the polynucleotides areincorporated onto a solid support, or gene chip. For the purposes of thepresent invention, a peptide nucleic acid (PNA) is a polyamide type ofDNA analog and the monomeric units for adenine, guanine, thymine andcytosine are available commercially (Perceptive Biosystems). Certaincomponents of DNA, such as phosphorus, phosphorus oxides, or deoxyribosederivatives, are not present in PNAs. As disclosed by P. E. Nielsen, M.Egholm, R. H. Berg and O. Buchardt, Science 254, 1497 (1991); and M.Egholm, O. Buchardt, L. Christensen, C. Behrens, S. M. Freier, D. A.Driver, R. H. Berg, S. K. Kim, B. Norden, and P. E. Nielsen, Nature 365,666 (1993), PNAs bind specifically and tightly to complementary DNAstrands and are not degraded by nucleases. In fact, PNA binds morestrongly to DNA than DNA itself does. This is probably because there isno electrostatic repulsion between the two strands, and also thepolyamide backbone is more flexible. Because of this, PNA/DNA duplexesbind under a wider range of stringency conditions than DNA/DNA duplexes,making it easier to perform multiplex hybridization. Smaller probes canbe used than with DNA due to the stronger binding characteristics ofPNA:DNA hybrids. In addition, it is more likely that single basemismatches can be determined with PNA/DNA hybridization because a singlemismatch in a PNA/DNA 15-mer lowers the melting point (T.sub.m) by8°-20° C., vs. 4°-16° C. for the DNA/DNA 15-mer duplex. Also, theabsence of charge groups in PNA means that hybridization can be done atlow ionic strengths and reduce possible interference by salt during theanalysis.

In addition to the foregoing, a polynucleotide can be used to controlgene expression through triple helix formation or antisense DNA or RNA.Antisense techniques are discussed, for example, in Okano, J. Neurochem.56: 560 (1991); “Oligodeoxynucleotides as Antisense Inhibitors of GeneExpression, CRC Press, Boca Raton, Fla. (1988). Triple helix formationis discussed in, for instance Lee et al., Nucleic Acids Research 6: 3073(1979); Cooney et al., Science 241: 456 (1988); and Dervan et al.,Science 251: 1360 (1991). Both methods rely on binding of thepolynucleotide to a complementary DNA or RNA. For these techniques,preferred polynucleotides are usually oligonucleotides 20 to 40 bases inlength and complementary to either the region of the gene involved intranscription (triple helix—see Lee et al., Nucl. Acids Res. 6:3073(1979); Cooney et al., Science 241:456 (1988); and Dervan et al.,Science 251:1360 (1991)) or to the mRNA itself (antisense—Okano, J.Neurochem. 56:560 (1991); Oligodeoxy-nucleotides as Antisense Inhibitorsof Gene Expression, CRC Press, Boca Raton, Fla. (1988).) Triple helixformation optimally results in a shut-off of RNA transcription from DNA,while antisense RNA hybridization blocks translation of an mRNA moleculeinto polypeptide. Both techniques are effective in model systems, andthe information disclosed herein can be used to design antisense ortriple helix polynucleotides in an effort to treat or prevent disease.

The present invention encompasses the addition of a nuclear localizationsignal, operably linked to the 5′ end, 3′ end, or any location therein,to any of the oligonucleotides, anti sense oligonucleotides, triplehelix oligonucleotides, ribozymes, PNA oligonucleotides, and/orpolynucleotides, of the present invention. See, for example, G. Cutrona,et al., Nat. Biotech., 18:300-303, (2000); which is hereby incorporatedherein by reference.

Polynucleotides of the present invention are also useful in genetherapy. One goal of gene therapy is to insert a normal gene into anorganism having a defective gene, in an effort to correct the geneticdefect. The polynucleotides disclosed in the present invention offer ameans of targeting such genetic defects in a highly accurate manner.Another goal is to insert a new gene that was not present in the hostgenome, thereby producing a new trait in the host cell. In one example,polynucleotide sequences of the present invention may be used toconstruct chimeric RNA/DNA oligonucleotides corresponding to saidsequences, specifically designed to induce host cell mismatch repairmechanisms in an organism upon systemic injection, for example(Bartlett, R. J., et al., Nat. Biotech, 18:615-622 (2000), which ishereby incorporated by reference herein in its entirety). Such RNA/DNAoligonucleotides could be designed to correct genetic defects in certainhost strains, and/or to introduce desired phenotypes in the host (e.g.,introduction of a specific polymorphism within an endogenous genecorresponding to a polynucleotide of the present invention that mayameliorate and/or prevent a disease symptom and/or disorder, etc.).Alternatively, the polynucleotide sequence of the present invention maybe used to construct duplex oligonucleotides corresponding to saidsequence, specifically designed to correct genetic defects in certainhost strains, and/or to introduce desired phenotypes into the host(e.g., introduction of a specific polymorphism within an endogenous genecorresponding to a polynucleotide of the present invention that mayameliorate and/or prevent a disease symptom and/or disorder, etc). Suchmethods of using duplex oligonucleotides are known in the art and areencompassed by the present invention (see EP1007712, which is herebyincorporated by reference herein in its entirety).

The polynucleotides are also useful for identifying organisms fromminute biological samples. The United States military, for example, isconsidering the use of restriction fragment length polymorphism (RFLP)for identification of its personnel. In this technique, an individual'sgenomic DNA is digested with one or more restriction enzymes, and probedon a Southern blot to yield unique bands for identifying personnel. Thismethod does not suffer from the current limitations of “Dog Tags” whichcan be lost, switched, or stolen, making positive identificationdifficult. The polynucleotides of the present invention can be used asadditional DNA markers for RFLP.

The polynucleotides of the present invention can also be used as analternative to RFLP, by determining the actual base-by-base DNA sequenceof selected portions of an organisms genome. These sequences can be usedto prepare PCR primers for amplifying and isolating such selected DNA,which can then be sequenced. Using this technique, organisms can beidentified because each organism will have a unique set of DNAsequences. Once an unique ID database is established for an organism,positive identification of that organism, living or dead, can be madefrom extremely small tissue samples. Similarly, polynucleotides of thepresent invention can be used as polymorphic markers, in addition to,the identification of transformed or non-transformed cells and/ortissues.

There is also a need for reagents capable of identifying the source of aparticular tissue. Such need arises, for example, when presented withtissue of unknown origin. Appropriate reagents can comprise, forexample, DNA probes or primers specific to particular tissue preparedfrom the sequences of the present invention. Panels of such reagents canidentify tissue by species and/or by organ type. In a similar fashion,these reagents can be used to screen tissue cultures for contamination.Moreover, as mentioned above, such reagents can be used to screen and/oridentify transformed and non-transformed cells and/or tissues.

In the very least, the polynucleotides of the present invention can beused as molecular weight markers on Southern gels, as diagnostic probesfor the presence of a specific mRNA in a particular cell type, as aprobe to “subtract-out” known sequences in the process of discoveringnovel polynucleotides, for selecting and making oligomers for attachmentto a “gene chip” or other support, to raise anti-DNA antibodies usingDNA immunization techniques, and as an antigen to elicit an immuneresponse.

Uses of the Polypeptides

Each of the polypeptides identified herein can be used in numerous ways.The following description should be considered exemplary and utilizesknown techniques.

A polypeptide of the present invention can be used to assay proteinlevels in a biological sample using antibody-based techniques. Forexample, protein expression in tissues can be studied with classicalimmunohistological methods. (Jalkanen, M., et al., J. Cell. Biol.101:976-985 (1985); Jalkanen, M., et al., J. Cell. Biol. 105:3087-3096(1987).) Other antibody-based methods useful for detecting protein geneexpression include immunoassays, such as the enzyme linked immunosorbentassay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assaylabels are known in the art and include enzyme labels, such as, glucoseoxidase, and radioisotopes, such as iodine (125I, 121I), carbon (14C),sulfur (35S), tritium (3H), indium (112In), and technetium (99mTc), andfluorescent labels, such as fluorescein and rhodamine, and biotin.

In addition to assaying protein levels in a biological sample, proteinscan also be detected in vivo by imaging. Antibody labels or markers forin vivo imaging of protein include those detectable by X-radiography,NMR or ESR. For X-radiography, suitable labels include radioisotopessuch as barium or cesium, which emit detectable radiation but are notovertly harmful to the subject. Suitable markers for NMR and ESR includethose with a detectable characteristic spin, such as deuterium, whichmay be incorporated into the antibody by labeling of nutrients for therelevant hybridoma.

A protein-specific antibody or antibody fragment which has been labeledwith an appropriate detectable imaging moiety, such as a radioisotope(for example, 131I, 112In, 99mTc), a radio-opaque substance, or amaterial detectable by nuclear magnetic resonance, is introduced (forexample, parenterally, subcutaneously, or intraperitoneally) into themammal. It will be understood in the art that the size of the subjectand the imaging system used will determine the quantity of imagingmoiety needed to produce diagnostic images. In the case of aradioisotope moiety, for a human subject, the quantity of radioactivityinjected will normally range from about 5 to 20 millicuries of 99mTc.The labeled antibody or antibody fragment will then preferentiallyaccumulate at the location of cells which contain the specific protein.In vivo tumor imaging is described in S. W. Burchiel et al.,“Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments.”(Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982).)

Thus, the invention provides a diagnostic method of a disorder, whichinvolves (a) assaying the expression of a polypeptide of the presentinvention in cells or body fluid of an individual; (b) comparing thelevel of gene expression with a standard gene expression level, wherebyan increase or decrease in the assayed polypeptide gene expression levelcompared to the standard expression level is indicative of a disorder.With respect to cancer, the presence of a relatively high amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Moreover, polypeptides of the present invention can be used to treat,prevent, and/or diagnose disease. For example, patients can beadministered a polypeptide of the present invention in an effort toreplace absent or decreased levels of the polypeptide (e.g., insulin),to supplement absent or decreased levels of a different polypeptide(e.g., hemoglobin S for hemoglobin B, SOD, catalase, DNA repairproteins), to inhibit the activity of a polypeptide (e.g., an oncogeneor tumor suppressor), to activate the activity of a polypeptide (e.g.,by binding to a receptor), to reduce the activity of a membrane boundreceptor by competing with it for free ligand (e.g., soluble TNFreceptors used in reducing inflammation), or to bring about a desiredresponse (e.g., blood vessel growth inhibition, enhancement of theimmune response to proliferative cells or tissues).

Similarly, antibodies directed to a polypeptide of the present inventioncan also be used to treat, prevent, and/or diagnose disease. Forexample, administration of an antibody directed to a polypeptide of thepresent invention can bind and reduce overproduction of the polypeptide.Similarly, administration of an antibody can activate the polypeptide,such as by binding to a polypeptide bound to a membrane (receptor).

At the very least, the polypeptides of the present invention can be usedas molecular weight markers on SDS-PAGE gels or on molecular sieve gelfiltration columns using methods well known to those of skill in theart. Polypeptides can also be used to raise antibodies, which in turnare used to measure protein expression from a recombinant cell, as a wayof assessing transformation of the host cell. Moreover, the polypeptidesof the present invention can be used to test the following biologicalactivities.

Gene Therapy Methods

Another aspect of the present invention is to gene therapy methods fortreating or preventing disorders, diseases and conditions. The genetherapy methods relate to the introduction of nucleic acid (DNA, RNA andantisense DNA or RNA) sequences into an animal to achieve expression ofa polypeptide of the present invention. This method requires apolynucleotide which codes for a polypeptide of the invention thatoperatively linked to a promoter and any other genetic elementsnecessary for the expression of the polypeptide by the target tissue.Such gene therapy and delivery techniques are known in the art, see, forexample, WO90/11092, which is herein incorporated by reference.

Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) comprising a promoter operably linked to apolynucleotide of the invention ex vivo, with the engineered cells thenbeing provided to a patient to be treated with the polypeptide. Suchmethods are well-known in the art. For example, see Belldegrun et al.,J. Natl Cancer Inst., 85:207-216 (1993); Ferrantini et al., CancerResearch, 53:107-1112 (1993); Ferrantini et al., J. Immunology 153:4604-4615 (1994); Kaido, T., et al., Int. J. Cancer 60: 221-229 (1995);Ogura et al., Cancer Research 50: 5102-5106 (1990); Santodonato, et al.,Human Gene Therapy 7:1-10 (1996); Santodonato, et al., Gene Therapy4:1246-1255 (1997); and Zhang, et al., Cancer Gene Therapy 3: 31-38(1996)), which are herein incorporated by reference. In one embodiment,the cells which are engineered are arterial cells. The arterial cellsmay be reintroduced into the patient through direct injection to theartery, the tissues surrounding the artery, or through catheterinjection.

As discussed in more detail below, the polynucleotide constructs can bedelivered by any method that delivers injectable materials to the cellsof an animal, such as, injection into the interstitial space of tissues(heart, muscle, skin, lung, liver, and the like). The polynucleotideconstructs may be delivered in a pharmaceutically acceptable liquid oraqueous carrier.

In one embodiment, the polynucleotide of the invention is delivered as anaked polynucleotide. The term “naked” polynucleotide, DNA or RNA refersto sequences that are free from any delivery vehicle that acts toassist, promote or facilitate entry into the cell, including viralsequences, viral particles, liposome formulations, lipofectin orprecipitating agents and the like. However, the polynucleotides of theinvention can also be delivered in liposome formulations and lipofectinformulations and the like can be prepared by methods well known to thoseskilled in the art. Such methods are described, for example, in U.S.Pat. Nos. 5,593,972, 5,589,466, and 5,580,859, which are hereinincorporated by reference.

The polynucleotide vector constructs of the invention used in the genetherapy method are preferably constructs that will not integrate intothe host genome nor will they contain sequences that allow forreplication. Appropriate vectors include pWLNEO, pSV2CAT, pOG44, pXT1and pSG available from Stratagene; pSVK3, pBPV, pMSG and pSVL availablefrom Pharmacia; and pEF1/V5, pcDNA3.1, and pRc/CMV2 available fromInvitrogen. Other suitable vectors will be readily apparent to theskilled artisan.

Any strong promoter known to those skilled in the art can be used fordriving the expression of polynucleotide sequence of the invention.Suitable promoters include adenoviral promoters, such as the adenoviralmajor late promoter; or heterologous promoters, such as thecytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV)promoter; inducible promoters, such as the MMT promoter, themetallothionein promoter; heat shock promoters; the albumin promoter;the ApoAI promoter; human globin promoters; viral thymidine kinasepromoters, such as the Herpes Simplex thymidine kinase promoter;retroviral LTRs; the b-actin promoter; and human growth hormonepromoters. The promoter also may be the native promoter for thepolynucleotides of the invention.

Unlike other gene therapy techniques, one major advantage of introducingnaked nucleic acid sequences into target cells is the transitory natureof the polynucleotide synthesis in the cells. Studies have shown thatnon-replicating DNA sequences can be introduced into cells to provideproduction of the desired polypeptide for periods of up to six months.

The polynucleotide construct of the invention can be delivered to theinterstitial space of tissues within the an animal, including of muscle,skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph,blood, bone, cartilage, pancreas, kidney, gall bladder, stomach,intestine, testis, ovary, uterus, rectum, nervous system, eye, gland,and connective tissue. Interstitial space of the tissues comprises theintercellular, fluid, mucopolysaccharide matrix among the reticularfibers of organ tissues, elastic fibers in the walls of vessels orchambers, collagen fibers of fibrous tissues, or that same matrix withinconnective tissue ensheathing muscle cells or in the lacunae of bone. Itis similarly the space occupied by the plasma of the circulation and thelymph fluid of the lymphatic channels. Delivery to the interstitialspace of muscle tissue is preferred for the reasons discussed below.They may be conveniently delivered by injection into the tissuescomprising these cells. They are preferably delivered to and expressedin persistent, non-dividing cells which are differentiated, althoughdelivery and expression may be achieved in non-differentiated or lesscompletely differentiated cells, such as, for example, stem cells ofblood or skin fibroblasts. In vivo muscle cells are particularlycompetent in their ability to take up and express polynucleotides.

For the naked nucleic acid sequence injection, an effective dosageamount of DNA or RNA will be in the range of from about 0.05 mg/kg bodyweight to about 50 mg/kg body weight. Preferably the dosage will be fromabout 0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05mg/kg to about 5 mg/kg. Of course, as the artisan of ordinary skill willappreciate, this dosage will vary according to the tissue site ofinjection. The appropriate and effective dosage of nucleic acid sequencecan readily be determined by those of ordinary skill in the art and maydepend on the condition being treated and the route of administration.

The preferred route of administration is by the parenteral route ofinjection into the interstitial space of tissues. However, otherparenteral routes may also be used, such as, inhalation of an aerosolformulation particularly for delivery to lungs or bronchial tissues,throat or mucous membranes of the nose. In addition, naked DNAconstructs can be delivered to arteries during angioplasty by thecatheter used in the procedure.

The naked polynucleotides are delivered by any method known in the art,including, but not limited to, direct needle injection at the deliverysite, intravenous injection, topical administration, catheter infusion,and so-called “gene guns”. These delivery methods are known in the art.

The constructs may also be delivered with delivery vehicles such asviral sequences, viral particles, liposome formulations, lipofectin,precipitating agents, etc. Such methods of delivery are known in theart.

In certain embodiments, the polynucleotide constructs of the inventionare complexed in a liposome preparation. Liposomal preparations for usein the instant invention include cationic (positively charged), anionic(negatively charged) and neutral preparations. However, cationicliposomes are particularly preferred because a tight charge complex canbe formed between the cationic liposome and the polyanionic nucleicacid. Cationic liposomes have been shown to mediate intracellulardelivery of plasmid DNA (Feigner et al., Proc. Natl. Acad. Sci. USA,84:7413-7416 (1987), which is herein incorporated by reference); mRNA(Malone et al., Proc. Natl. Acad. Sci. USA, 86:6077-6081 (1989), whichis herein incorporated by reference); and purified transcription factors(Debs et al., J. Biol. Chem., 265:10189-10192 (1990), which is hereinincorporated by reference), in functional form.

Cationic liposomes are readily available. For example,N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes areparticularly useful and are available under the trademark Lipofectin,from GIBCO BRL, Grand Island, N.Y. (See, also, Feigner et al., Proc.Natl. Acad. Sci. USA, 84:7413-7416 (1987), which is herein incorporatedby reference). Other commercially available liposomes includetransfectace (DDAB/DOPE) and DOTAP/DOPE (Boehringer).

Other cationic liposomes can be prepared from readily availablematerials using techniques well known in the art. See, e.g. PCTPublication NO: WO 90/11092 (which is herein incorporated by reference)for a description of the synthesis of DOTAP(1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes. Preparationof DOTMA liposomes is explained in the literature, see, e.g., Feigner etal., Proc. Natl. Acad. Sci. USA, 84:7413-7417, which is hereinincorporated by reference. Similar methods can be used to prepareliposomes from other cationic lipid materials.

Similarly, anionic and neutral liposomes are readily available, such asfrom Avanti Polar Lipids (Birmingham, Ala.), or can be easily preparedusing readily available materials. Such materials include phosphatidyl,choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidyl glycerol (DOPG),dioleoylphoshatidyl ethanolamine (DOPE), among others. These materialscan also be mixed with the DOTMA and DOTAP starting materials inappropriate ratios. Methods for making liposomes using these materialsare well known in the art.

For example, commercially dioleoylphosphatidyl choline (DOPC),dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidylethanolamine (DOPE) can be used in various combinations to makeconventional liposomes, with or without the addition of cholesterol.Thus, for example, DOPG/DOPC vesicles can be prepared by drying 50 mgeach of DOPG and DOPC under a stream of nitrogen gas into a sonicationvial. The sample is placed under a vacuum pump overnight and is hydratedthe following day with deionized water. The sample is then sonicated for2 hours in a capped vial, using a Heat Systems model 350 sonicatorequipped with an inverted cup (bath type) probe at the maximum settingwhile the bath is circulated at 15EC. Alternatively, negatively chargedvesicles can be prepared without sonication to produce multilamellarvesicles or by extrusion through nucleopore membranes to produceunilamellar vesicles of discrete size. Other methods are known andavailable to those of skill in the art.

The liposomes can comprise multilamellar vesicles (MLVs), smallunilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs), withSUVs being preferred. The various liposome-nucleic acid complexes areprepared using methods well known in the art. See, e.g., Straubinger etal., Methods of Immunology, 101:512-527 (1983), which is hereinincorporated by reference. For example, MLVs containing nucleic acid canbe prepared by depositing a thin film of phospholipid on the walls of aglass tube and subsequently hydrating with a solution of the material tobe encapsulated. SUVs are prepared by extended sonication of MLVs toproduce a homogeneous population of unilamellar liposomes. The materialto be entrapped is added to a suspension of preformed MLVs and thensonicated. When using liposomes containing cationic lipids, the driedlipid film is resuspended in an appropriate solution such as sterilewater or an isotonic buffer solution such as 10 mM Tris/NaCl, sonicated,and then the preformed liposomes are mixed directly with the DNA. Theliposome and DNA form a very stable complex due to binding of thepositively charged liposomes to the cationic DNA. SUVs find use withsmall nucleic acid fragments. LUVs are prepared by a number of methods,well known in the art. Commonly used methods include Ca2+-EDTA chelation(Papahadjopoulos et al., Biochim. Biophys. Acta, 394:483 (1975); Wilsonet al., Cell, 17:77 (1979)); ether injection (Deamer et al., Biochim.Biophys. Acta, 443:629 (1976); Ostro et al., Biochem. Biophys. Res.Commun., 76:836 (1977); Fraley et al., Proc. Natl. Acad. Sci. USA,76:3348 (1979)); detergent dialysis (Enoch et al., Proc. Natl. Acad.Sci. USA, 76:145 (1979)); and reverse-phase evaporation (REV) (Fraley etal., J. Biol. Chem. 255:10431 (1980); Szoka et al., Proc. Natl. Acad.Sci. USA, 75:145 (1978); Schaefer-Ridder et al., Science, 215:166(1982)), which are herein incorporated by reference.

Generally, the ratio of DNA to liposomes will be from about 10:1 toabout 1:10. Preferably, the ration will be from about 5:1 to about 1:5.More preferably, the ration will be about 3:1 to about 1:3. Still morepreferably, the ratio will be about 1:1.

U.S. Pat. No. 5,676,954 (which is herein incorporated by reference)reports on the injection of genetic material, complexed with cationicliposomes carriers, into mice. U.S. Pat. Nos. 4,897,355, 4,946,787,5,049,386, 5,459,127, 5,589,466, 5,693,622, 5,580,859, 5,703,055, andinternational publication NO: WO 94/9469 (which are herein incorporatedby reference) provide cationic lipids for use in transfecting DNA intocells and mammals. U.S. Pat. Nos. 5,589,466, 5,693,622, 5,580,859,5,703,055, and international publication NO: WO 94/9469 (which areherein incorporated by reference) provide methods for deliveringDNA-cationic lipid complexes to mammals.

In certain embodiments, cells are engineered, ex vivo or in vivo, usinga retroviral particle containing RNA which comprises a sequence encodingpolypeptides of the invention. Retroviruses from which the retroviralplasmid vectors may be derived include, but are not limited to, MoloneyMurine Leukemia Virus, spleen necrosis virus, Rous sarcoma Virus, HarveySarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, humanimmunodeficiency virus, Myeloproliferative Sarcoma Virus, and mammarytumor virus.

The retroviral plasmid vector is employed to transduce packaging celllines to form producer cell lines. Examples of packaging cells which maybe transfected include, but are not limited to, the PE501, PA317, R-2,R-AM, PA12, T19-14X, VT-19-17-H2, RCRE, RCRIP, GP+E-86, GP+envAm12, andDAN cell lines as described in Miller, Human Gene Therapy, 1:5-14(1990), which is incorporated herein by reference in its entirety. Thevector may transduce the packaging cells through any means known in theart. Such means include, but are not limited to, electroporation, theuse of liposomes, and CaPO4 precipitation. In one alternative, theretroviral plasmid vector may be encapsulated into a liposome, orcoupled to a lipid, and then administered to a host.

The producer cell line generates infectious retroviral vector particleswhich include polynucleotide encoding polypeptides of the invention.Such retroviral vector particles then may be employed, to transduceeukaryotic cells, either in vitro or in vivo. The transduced eukaryoticcells will express polypeptides of the invention.

In certain other embodiments, cells are engineered, ex vivo or in vivo,with polynucleotides of the invention contained in an adenovirus vector.Adenovirus can be manipulated such that it encodes and expressespolypeptides of the invention, and at the same time is inactivated interms of its ability to replicate in a normal lytic viral life cycle.Adenovirus expression is achieved without integration of the viral DNAinto the host cell chromosome, thereby alleviating concerns aboutinsertional mutagenesis. Furthermore, adenoviruses have been used aslive enteric vaccines for many years with an excellent safety profile(Schwartz et al., Am. Rev. Respir. Dis., 109:233-238 (1974)). Finally,adenovirus mediated gene transfer has been demonstrated in a number ofinstances including transfer of alpha-1-antitrypsin and CFTR to thelungs of cotton rats (Rosenfeld et al., Science, 252:431-434 (1991);Rosenfeld et al., Cell, 68:143-155 (1992)). Furthermore, extensivestudies to attempt to establish adenovirus as a causative agent in humancancer were uniformly negative (Green et al. Proc. Natl. Acad. Sci. USA,76:6606 (1979)).

Suitable adenoviral vectors useful in the present invention aredescribed, for example, in Kozarsky and Wilson, Curr. Opin. Genet.Devel., 3:499-503 (1993); Rosenfeld et al., Cell, 68:143-155 (1992);Engelhardt et al., Human Genet. Ther., 4:759-769 (1993); Yang et al.,Nature Genet., 7:362-369 (1994); Wilson et al., Nature, 365:691-692(1993); and U.S. Pat. No. 5,652,224, which are herein incorporated byreference. For example, the adenovirus vector Ad2 is useful and can begrown in human 293 cells. These cells contain the E1 region ofadenovirus and constitutively express E1a and E1b, which complement thedefective adenoviruses by providing the products of the genes deletedfrom the vector. In addition to Ad2, other varieties of adenovirus(e.g., Ad3, Ad5, and Ad7) are also useful in the present invention.

Preferably, the adenoviruses used in the present invention arereplication deficient. Replication deficient adenoviruses require theaid of a helper virus and/or packaging cell line to form infectiousparticles. The resulting virus is capable of infecting cells and canexpress a polynucleotide of interest which is operably linked to apromoter, but cannot replicate in most cells. Replication deficientadenoviruses may be deleted in one or more of all or a portion of thefollowing genes: E1a, E1b, E3, E4, E2a, or L1 through L5.

In certain other embodiments, the cells are engineered, ex vivo or invivo, using an adeno-associated virus (AAV). AAVs are naturallyoccurring defective viruses that require helper viruses to produceinfectious particles (Muzyczka, Curr. Topics in Microbiol. Immunol.,158:97 (1992)). It is also one of the few viruses that may integrate itsDNA into non-dividing cells. Vectors containing as little as 300 basepairs of AAV can be packaged and can integrate, but space for exogenousDNA is limited to about 4.5 kb. Methods for producing and using suchAAVs are known in the art. See, for example, U.S. Pat. Nos. 5,139,941,5,173,414, 5,354,678, 5,436,146, 5,474,935, 5,478,745, and 5,589,377.

For example, an appropriate AAV vector for use in the present inventionwill include all the sequences necessary for DNA replication,encapsidation, and host-cell integration. The polynucleotide constructcontaining polynucleotides of the invention is inserted into the AAVvector using standard cloning methods, such as those found in Sambrooket al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press(1989). The recombinant AAV vector is then transfected into packagingcells which are infected with a helper virus, using any standardtechnique, including lipofection, electroporation, calcium phosphateprecipitation, etc. Appropriate helper viruses include adenoviruses,cytomegaloviruses, vaccinia viruses, or herpes viruses. Once thepackaging cells are transfected and infected, they will produceinfectious AAV viral particles which contain the polynucleotideconstruct of the invention. These viral particles are then used totransduce eukaryotic cells, either ex vivo or in vivo. The transducedcells will contain the polynucleotide construct integrated into itsgenome, and will express the desired gene product.

Another method of gene therapy involves operably associatingheterologous control regions and endogenous polynucleotide sequences(e.g. encoding the polypeptide sequence of interest) via homologousrecombination (see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997;International Publication NO: WO 96/29411, published Sep. 26, 1996;International Publication NO: WO 94/12650, published Aug. 4, 1994;Koller et al., Proc. Natl. Acad. Sci. USA, 86:8932-8935 (1989); andZijlstra et al., Nature, 342:435-438 (1989). This method involves theactivation of a gene which is present in the target cells, but which isnot normally expressed in the cells, or is expressed at a lower levelthan desired.

Polynucleotide constructs are made, using standard techniques known inthe art, which contain the promoter with targeting sequences flankingthe promoter. Suitable promoters are described herein. The targetingsequence is sufficiently complementary to an endogenous sequence topermit homologous recombination of the promoter-targeting sequence withthe endogenous sequence. The targeting sequence will be sufficientlynear the 5′ end of the desired endogenous polynucleotide sequence so thepromoter will be operably linked to the endogenous sequence uponhomologous recombination.

The promoter and the targeting sequences can be amplified using PCR.Preferably, the amplified promoter contains distinct restriction enzymesites on the 5′ and 3′ ends. Preferably, the 3′ end of the firsttargeting sequence contains the same restriction enzyme site as the 5′end of the amplified promoter and the 5′ end of the second targetingsequence contains the same restriction site as the 3′ end of theamplified promoter. The amplified promoter and targeting sequences aredigested and ligated together.

The promoter-targeting sequence construct is delivered to the cells,either as naked polynucleotide, or in conjunction withtransfection-facilitating agents, such as liposomes, viral sequences,viral particles, whole viruses, lipofection, precipitating agents, etc.,described in more detail above. The P promoter-targeting sequence can bedelivered by any method, included direct needle injection, intravenousinjection, topical administration, catheter infusion, particleaccelerators, etc. The methods are described in more detail below.

The promoter-targeting sequence construct is taken up by cells.Homologous recombination between the construct and the endogenoussequence takes place, such that an endogenous sequence is placed underthe control of the promoter. The promoter then drives the expression ofthe endogenous sequence.

The polynucleotides encoding polypeptides of the present invention maybe administered along with other polynucleotides encoding angiogenicproteins. Angiogenic proteins include, but are not limited to, acidicand basic fibroblast growth factors, VEGF-1, VEGF-2 (VEGF-C), VEGF-3(VEGF-B), epidermal growth factor alpha and beta, platelet-derivedendothelial cell growth factor, platelet-derived growth factor, tumornecrosis factor alpha, hepatocyte growth factor, insulin like growthfactor, colony stimulating factor, macrophage colony stimulating factor,granulocyte/macrophage colony stimulating factor, and nitric oxidesynthase.

Preferably, the polynucleotide encoding a polypeptide of the inventioncontains a secretory signal sequence that facilitates secretion of theprotein. Typically, the signal sequence is positioned in the codingregion of the polynucleotide to be expressed towards or at the 5′ end ofthe coding region. The signal sequence may be homologous or heterologousto the polynucleotide of interest and may be homologous or heterologousto the cells to be transfected. Additionally, the signal sequence may bechemically synthesized using methods known in the art.

Any mode of administration of any of the above-described polynucleotidesconstructs can be used so long as the mode results in the expression ofone or more molecules in an amount sufficient to provide a therapeuticeffect. This includes direct needle injection, systemic injection,catheter infusion, biolistic injectors, particle accelerators (i.e.,“gene guns”), gelfoam sponge depots, other commercially available depotmaterials, osmotic pumps (e.g., Alza minipumps), oral or suppositorialsolid (tablet or pill) pharmaceutical formulations, and decanting ortopical applications during surgery. For example, direct injection ofnaked calcium phosphate-precipitated plasmid into rat liver and ratspleen or a protein-coated plasmid into the portal vein has resulted ingene expression of the foreign gene in the rat livers. (Kaneda et al.,Science, 243:375 (1989)).

A preferred method of local administration is by direct injection.Preferably, a recombinant molecule of the present invention complexedwith a delivery vehicle is administered by direct injection into orlocally within the area of arteries. Administration of a compositionlocally within the area of arteries refers to injecting the compositioncentimeters and preferably, millimeters within arteries.

Another method of local administration is to contact a polynucleotideconstruct of the present invention in or around a surgical wound. Forexample, a patient can undergo surgery and the polynucleotide constructcan be coated on the surface of tissue inside the wound or the constructcan be injected into areas of tissue inside the wound.

Therapeutic compositions useful in systemic administration, includerecombinant molecules of the present invention complexed to a targeteddelivery vehicle of the present invention. Suitable delivery vehiclesfor use with systemic administration comprise liposomes comprisingligands for targeting the vehicle to a particular site.

Preferred methods of systemic administration, include intravenousinjection, aerosol, oral and percutaneous (topical) delivery.Intravenous injections can be performed using methods standard in theart. Aerosol delivery can also be performed using methods standard inthe art (see, for example, Stribling et al., Proc. Natl. Acad. Sci. USA,189:11277-11281 (1992), which is incorporated herein by reference). Oraldelivery can be performed by complexing a polynucleotide construct ofthe present invention to a carrier capable of withstanding degradationby digestive enzymes in the gut of an animal. Examples of such carriers,include plastic capsules or tablets, such as those known in the art.Topical delivery can be performed by mixing a polynucleotide constructof the present invention with a lipophilic reagent (e.g., DMSO) that iscapable of passing into the skin.

Determining an effective amount of substance to be delivered can dependupon a number of factors including, for example, the chemical structureand biological activity of the substance, the age and weight of theanimal, the precise condition requiring treatment and its severity, andthe route of administration. The frequency of treatments depends upon anumber of factors, such as the amount of polynucleotide constructsadministered per dose, as well as the health and history of the subject.The precise amount, number of doses, and timing of doses will bedetermined by the attending physician or veterinarian. Therapeuticcompositions of the present invention can be administered to any animal,preferably to mammals and birds. Preferred mammals include humans, dogs,cats, mice, rats, rabbits sheep, cattle, horses and pigs, with humansbeing particularly preferred.

Biological Activities

The polynucleotides or polypeptides, or agonists or antagonists of thepresent invention can be used in assays to test for one or morebiological activities. If these polynucleotides and polypeptides doexhibit activity in a particular assay, it is likely that thesemolecules may be involved in the diseases associated with the biologicalactivity. Thus, the polynucleotides or polypeptides, or agonists orantagonists could be used to treat the associated disease.

Immune Activity

The polynucleotides or polypeptides, or agonists or antagonists of thepresent invention may be useful in treating, preventing, and/ordiagnosing diseases, disorders, and/or conditions of the immune system,by activating or inhibiting the proliferation, differentiation, ormobilization (chemotaxis) of immune cells. Immune cells develop througha process called hematopoiesis, producing myeloid (platelets, red bloodcells, neutrophils, and macrophages) and lymphoid (B and T lymphocytes)cells from pluripotent stem cells. The etiology of these immunediseases, disorders, and/or conditions may be genetic, somatic, such ascancer or some autoimmune diseases, disorders, and/or conditions,acquired (e.g., by chemotherapy or toxins), or infectious. Moreover, apolynucleotides or polypeptides, or agonists or antagonists of thepresent invention can be used as a marker or detector of a particularimmune system disease or disorder.

A polynucleotides or polypeptides, or agonists or antagonists of thepresent invention may be useful in treating, preventing, and/ordiagnosing diseases, disorders, and/or conditions of hematopoieticcells. A polynucleotides or polypeptides, or agonists or antagonists ofthe present invention could be used to increase differentiation andproliferation of hematopoietic cells, including the pluripotent stemcells, in an effort to treat or prevent those diseases, disorders,and/or conditions associated with a decrease in certain (or many) typeshematopoietic cells. Examples of immunologic deficiency syndromesinclude, but are not limited to: blood protein diseases, disorders,and/or conditions (e.g. agammaglobulinemia, dysgammaglobulinemia),ataxia telangiectasia, common variable immunodeficiency, DigeorgeSyndrome, HIV infection, HTLV-BLV infection, leukocyte adhesiondeficiency syndrome, lymphopenia, phagocyte bactericidal dysfunction,severe combined immunodeficiency (SCIDs), Wiskott-Aldrich Disorder,anemia, thrombocytopenia, or hemoglobinuria.

Moreover, a polynucleotides or polypeptides, or agonists or antagonistsof the present invention could also be used to modulate hemostatic (thestopping of bleeding) or thrombolytic activity (clot formation). Forexample, by increasing hemostatic or thrombolytic activity, apolynucleotides or polypeptides, or agonists or antagonists of thepresent invention could be used to treat or prevent blood coagulationdiseases, disorders, and/or conditions (e.g., afibrinogenemia, factordeficiencies, arterial thrombosis, venous thrombosis, etc.), bloodplatelet diseases, disorders, and/or conditions (e.g. thrombocytopenia),or wounds resulting from trauma, surgery, or other causes.Alternatively, a polynucleotides or polypeptides, or agonists orantagonists of the present invention that can decrease hemostatic orthrombolytic activity could be used to inhibit or dissolve clotting.Polynucleotides or polypeptides, or agonists or antagonists of thepresent invention are may also be useful for the detection, prognosis,treatment, and/or prevention of heart attacks (infarction), strokes,scarring, fibrinolysis, uncontrolled bleeding, uncontrolled coagulation,uncontrolled complement fixation, and/or inflammation.

A polynucleotides or polypeptides, or agonists or antagonists of thepresent invention may also be useful in treating, preventing, and/ordiagnosing autoimmune diseases, disorders, and/or conditions. Manyautoimmune diseases, disorders, and/or conditions result frominappropriate recognition of self as foreign material by immune cells.This inappropriate recognition results in an immune response leading tothe destruction of the host tissue. Therefore, the administration of apolynucleotides or polypeptides, or agonists or antagonists of thepresent invention that inhibits an immune response, particularly theproliferation, differentiation, or chemotaxis of T-cells, may be aneffective therapy in preventing autoimmune diseases, disorders, and/orconditions.

Examples of autoimmune diseases, disorders, and/or conditions that canbe treated, prevented, and/or diagnosed or detected by the presentinvention include, but are not limited to: Addison's Disease, hemolyticanemia, antiphospholipid syndrome, rheumatoid arthritis, dermatitis,allergic encephalomyelitis, glomerulonephritis, Goodpasture's Syndrome,Graves' Disease, Multiple Sclerosis, Myasthenia Gravis, Neuritis,Ophthalmia, Bullous Pemphigoid, Pemphigus, Polyendocrinopathies,Purpura, Reiter's Disease, Stiff-Man Syndrome, Autoimmune Thyroiditis,Systemic Lupus Erythematosus, Autoimmune Pulmonary Inflammation,Guillain-Barre Syndrome, insulin dependent diabetes mellitis, andautoimmune inflammatory eye disease.

Similarly, allergic reactions and conditions, such as asthma(particularly allergic asthma) or other respiratory problems, may alsobe treated, prevented, and/or diagnosed by polynucleotides orpolypeptides, or agonists or antagonists of the present invention.Moreover, these molecules can be used to treat anaphylaxis,hypersensitivity to an antigenic molecule, or blood groupincompatibility.

A polynucleotides or polypeptides, or agonists or antagonists of thepresent invention may also be used to treat, prevent, and/or diagnoseorgan rejection or graft-versus-host disease (GVHD). Organ rejectionoccurs by host immune cell destruction of the transplanted tissuethrough an immune response. Similarly, an immune response is alsoinvolved in GVHD, but, in this case, the foreign transplanted immunecells destroy the host tissues. The administration of a polynucleotidesor polypeptides, or agonists or antagonists of the present inventionthat inhibits an immune response, particularly the proliferation,differentiation, or chemotaxis of T-cells, may be an effective therapyin preventing organ rejection or GVHD.

Similarly, a polynucleotides or polypeptides, or agonists or antagonistsof the present invention may also be used to modulate inflammation. Forexample, the polypeptide or polynucleotide or agonists or antagonist mayinhibit the proliferation and differentiation of cells involved in aninflammatory response. These molecules can be used to treat, prevent,and/or diagnose inflammatory conditions, both chronic and acuteconditions, including chronic prostatitis, granulomatous prostatitis andmalacoplakia, inflammation associated with infection (e.g., septicshock, sepsis, or systemic inflammatory response syndrome (SIRS)),ischemia-reperfusion injury, endotoxin lethality, arthritis,complement-mediated hyperacute rejection, nephritis, cytokine orchemokine induced lung injury, inflammatory bowel disease, Crohn'sdisease, or resulting from over production of cytokines (e.g., TNF orIL-1.)

Hyperproliferative Disorders

A polynucleotides or polypeptides, or agonists or antagonists of theinvention can be used to treat, prevent, and/or diagnosehyperproliferative diseases, disorders, and/or conditions, includingneoplasms. A polynucleotides or polypeptides, or agonists or antagonistsof the present invention may inhibit the proliferation of the disorderthrough direct or indirect interactions. Alternatively, apolynucleotides or polypeptides, or agonists or antagonists of thepresent invention may proliferate other cells which can inhibit thehyperproliferative disorder.

For example, by increasing an immune response, particularly increasingantigenic qualities of the hyperproliferative disorder or byproliferating, differentiating, or mobilizing T-cells,hyperproliferative diseases, disorders, and/or conditions can betreated, prevented, and/or diagnosed. This immune response may beincreased by either enhancing an existing immune response, or byinitiating a new immune response. Alternatively, decreasing an immuneresponse may also be a method of treating, preventing, and/or diagnosinghyperproliferative diseases, disorders, and/or conditions, such as achemotherapeutic agent.

Examples of hyperproliferative diseases, disorders, and/or conditionsthat can be treated, prevented, and/or diagnosed by polynucleotides orpolypeptides, or agonists or antagonists of the present inventioninclude, but are not limited to neoplasms located in the: colon,abdomen, bone, breast, digestive system, liver, pancreas, peritoneum,endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary,thymus, thyroid), eye, head and neck, nervous (central and peripheral),lymphatic system, pelvic, skin, soft tissue, spleen, thoracic, andurogenital.

Similarly, other hyperproliferative diseases, disorders, and/orconditions can also be treated, prevented, and/or diagnosed by apolynucleotides or polypeptides, or agonists or antagonists of thepresent invention. Examples of such hyperproliferative diseases,disorders, and/or conditions include, but are not limited to:hypergammaglobulinemia, lymphoproliferative diseases, disorders, and/orconditions, paraproteinemias, purpura, sarcoidosis, Sezary Syndrome,Waldenstron's Macroglobulinemia, Gaucher's Disease, histiocytosis, andany other hyperproliferative disease, besides neoplasia, located in anorgan system listed above.

One preferred embodiment utilizes polynucleotides of the presentinvention to inhibit aberrant cellular division, by gene therapy usingthe present invention, and/or protein fusions or fragments thereof.

Thus, the present invention provides a method for treating or preventingcell proliferative diseases, disorders, and/or conditions by insertinginto an abnormally proliferating cell a polynucleotide of the presentinvention, wherein said polynucleotide represses said expression.

Another embodiment of the present invention provides a method oftreating or preventing cell-proliferative diseases, disorders, and/orconditions in individuals comprising administration of one or moreactive gene copies of the present invention to an abnormallyproliferating cell or cells. In a preferred embodiment, polynucleotidesof the present invention is a DNA construct comprising a recombinantexpression vector effective in expressing a DNA sequence encoding saidpolynucleotides. In another preferred embodiment of the presentinvention, the DNA construct encoding the polynucleotides of the presentinvention is inserted into cells to be treated utilizing a retrovirus,or more Preferably an adenoviral vector (See G J. Nabel, et. al., PNAS1999 96: 324-326, which is hereby incorporated by reference). In a mostpreferred embodiment, the viral vector is defective and will nottransform non-proliferating cells, only proliferating cells. Moreover,in a preferred embodiment, the polynucleotides of the present inventioninserted into proliferating cells either alone, or in combination withor fused to other polynucleotides, can then be modulated via an externalstimulus (i.e. magnetic, specific small molecule, chemical, or drugadministration, etc.), which acts upon the promoter upstream of saidpolynucleotides to induce expression of the encoded protein product. Assuch the beneficial therapeutic affect of the present invention may beexpressly modulated (i.e. to increase, decrease, or inhibit expressionof the present invention) based upon said external stimulus.

Polynucleotides of the present invention may be useful in repressingexpression of oncogenic genes or antigens. By “repressing expression ofthe oncogenic genes” is intended the suppression of the transcription ofthe gene, the degradation of the gene transcript (pre-message RNA), theinhibition of splicing, the destruction of the messenger RNA, theprevention of the post-translational modifications of the protein, thedestruction of the protein, or the inhibition of the normal function ofthe protein.

For local administration to abnormally proliferating cells,polynucleotides of the present invention may be administered by anymethod known to those of skill in the art including, but not limited totransfection, electroporation, microinjection of cells, or in vehiclessuch as liposomes, lipofectin, or as naked polynucleotides, or any othermethod described throughout the specification. The polynucleotide of thepresent invention may be delivered by known gene delivery systems suchas, but not limited to, retroviral vectors (Gilboa, J. Virology 44:845(1982); Hocke, Nature 320:275 (1986); Wilson, et al., Proc. Natl. Acad.Sci. U.S.A. 85:3014), vaccinia virus system (Chakrabarty et al., Mol.Cell Biol. 5:3403 (1985) or other efficient DNA delivery systems (Yateset al., Nature 313:812 (1985)) known to those skilled in the art. Thesereferences are exemplary only and are hereby incorporated by reference.In order to specifically deliver or transfect cells which are abnormallyproliferating and spare non-dividing cells, it is preferable to utilizea retrovirus, or adenoviral (as described in the art and elsewhereherein) delivery system known to those of skill in the art. Since hostDNA replication is required for retroviral DNA to integrate and theretrovirus will be unable to self replicate due to the lack of theretrovirus genes needed for its life cycle. Utilizing such a retroviraldelivery system for polynucleotides of the present invention will targetsaid gene and constructs to abnormally proliferating cells and willspare the non-dividing normal cells.

The polynucleotides of the present invention may be delivered directlyto cell proliferative disorder/disease sites in internal organs, bodycavities and the like by use of imaging devices used to guide aninjecting needle directly to the disease site. The polynucleotides ofthe present invention may also be administered to disease sites at thetime of surgical intervention.

By “cell proliferative disease” is meant any human or animal disease ordisorder, affecting any one or any combination of organs, cavities, orbody parts, which is characterized by single or multiple local abnormalproliferations of cells, groups of cells, or tissues, whether benign ormalignant.

Any amount of the polynucleotides of the present invention may beadministered as long as it has a biologically inhibiting effect on theproliferation of the treated cells. Moreover, it is possible toadminister more than one of the polynucleotide of the present inventionsimultaneously to the same site. By “biologically inhibiting” is meantpartial or total growth inhibition as well as decreases in the rate ofproliferation or growth of the cells. The biologically inhibitory dosemay be determined by assessing the effects of the polynucleotides of thepresent invention on target malignant or abnormally proliferating cellgrowth in tissue culture, tumor growth in animals and cell cultures, orany other method known to one of ordinary skill in the art.

The present invention is further directed to antibody-based therapieswhich involve administering of anti-polypeptides and anti-polynucleotideantibodies to a mammalian, preferably human, patient for treating,preventing, and/or diagnosing one or more of the described diseases,disorders, and/or conditions. Methods for producing anti-polypeptidesand anti-polynucleotide antibodies polyclonal and monoclonal antibodiesare described in detail elsewhere herein. Such antibodies may beprovided in pharmaceutically acceptable compositions as known in the artor as described herein.

A summary of the ways in which the antibodies of the present inventionmay be used therapeutically includes binding polynucleotides orpolypeptides of the present invention locally or systemically in thebody or by direct cytotoxicity of the antibody, e.g. as mediated bycomplement (CDC) or by effector cells (ADCC). Some of these approachesare described in more detail below. Armed with the teachings providedherein, one of ordinary skill in the art will know how to use theantibodies of the present invention for diagnostic, monitoring ortherapeutic purposes without undue experimentation.

In particular, the antibodies, fragments and derivatives of the presentinvention are useful for treating, preventing, and/or diagnosing asubject having or developing cell proliferative and/or differentiationdiseases, disorders, and/or conditions as described herein. Suchtreatment comprises administering a single or multiple doses of theantibody, or a fragment, derivative, or a conjugate thereof.

The antibodies of this invention may be advantageously utilized incombination with other monoclonal or chimeric antibodies, or withlymphokines or hematopoietic growth factors, for example, which serve toincrease the number or activity of effector cells which interact withthe antibodies.

It is preferred to use high affinity and/or potent in vivo inhibitingand/or neutralizing antibodies against polypeptides or polynucleotidesof the present invention, fragments or regions thereof, for bothimmunoassays directed to and therapy of diseases, disorders, and/orconditions related to polynucleotides or polypeptides, includingfragments thereof, of the present invention. Such antibodies, fragments,or regions, will preferably have an affinity for polynucleotides orpolypeptides, including fragments thereof. Preferred binding affinitiesinclude those with a dissociation constant or Kd less than 5×10-6M,10-6M, 5×10-7M, 10-7M, 5×10-8M, 10-8M, 5×10-9M, 10-9M, 5×10-10M, 10-10M,5×10-11M, 10-11M, 5×10-12M, 10-12M, 5×10-13M, 10-13M, 5×10-14M, 10-14M,5×10-15M, and 10-15M.

Moreover, polypeptides of the present invention may be useful ininhibiting the angiogenesis of proliferative cells or tissues, eitheralone, as a protein fusion, or in combination with other polypeptidesdirectly or indirectly, as described elsewhere herein. In a mostpreferred embodiment, said anti-angiogenesis effect may be achievedindirectly, for example, through the inhibition of hematopoietic,tumor-specific cells, such as tumor-associated macrophages (See Joseph IB, et al. J Natl Cancer Inst, 90(21):1648-53 (1998), which is herebyincorporated by reference). Antibodies directed to polypeptides orpolynucleotides of the present invention may also result in inhibitionof angiogenesis directly, or indirectly (See Witte L, et al., CancerMetastasis Rev. 17(2):155-61 (1998), which is hereby incorporated byreference)).

Polypeptides, including protein fusions, of the present invention, orfragments thereof may be useful in inhibiting proliferative cells ortissues through the induction of apoptosis. Said polypeptides may acteither directly, or indirectly to induce apoptosis of proliferativecells and tissues, for example in the activation of a death-domainreceptor, such as tumor necrosis factor (TNF) receptor-1, CD95(Fas/APO-1), TNF-receptor-related apoptosis-mediated protein (TRAMP) andTNF-related apoptosis-inducing ligand (TRAIL) receptor-1 and -2 (SeeSchulze-Osthoff K, et al., Eur J Biochem 254(3):439-59 (1998), which ishereby incorporated by reference). Moreover, in another preferredembodiment of the present invention, said polypeptides may induceapoptosis through other mechanisms, such as in the activation of otherproteins which will activate apoptosis, or through stimulating theexpression of said proteins, either alone or in combination with smallmolecule drugs or adjuvants, such as apoptonin, galectins, thioredoxins,antiinflammatory proteins (See for example, Mutat. Res. 400(1-2):447-55(1998), Med Hypotheses. 50(5):423-33 (1998), Chem. Biol. Interact. April24; 111-112:23-34 (1998), J Mol Med. 76(6):402-12 (1998), Int. J. TissueReact. 20(1):3-15 (1998), which are all hereby incorporated byreference).

Polypeptides, including protein fusions to, or fragments thereof, of thepresent invention are useful in inhibiting the metastasis ofproliferative cells or tissues. Inhibition may occur as a direct resultof administering polypeptides, or antibodies directed to saidpolypeptides as described elsewhere herein, or indirectly, such asactivating the expression of proteins known to inhibit metastasis, forexample alpha 4 integrins, (See, e.g., Curr Top Microbiol Immunol 1998;231:125-41, which is hereby incorporated by reference). Such therapeuticaffects of the present invention may be achieved either alone, or incombination with small molecule drugs or adjuvants.

In another embodiment, the invention provides a method of deliveringcompositions containing the polypeptides of the invention (e.g.,compositions containing polypeptides or polypeptide antibodiesassociated with heterologous polypeptides, heterologous nucleic acids,toxins, or prodrugs) to targeted cells expressing the polypeptide of thepresent invention. Polypeptides or polypeptide antibodies of theinvention may be associated with heterologous polypeptides, heterologousnucleic acids, toxins, or prodrugs via hydrophobic, hydrophilic, ionicand/or covalent interactions.

Polypeptides, protein fusions to, or fragments thereof, of the presentinvention are useful in enhancing the immunogenicity and/or antigenicityof proliferating cells or tissues, either directly, such as would occurif the polypeptides of the present invention ‘vaccinated’ the immuneresponse to respond to proliferative antigens and immunogens, orindirectly, such as in activating the expression of proteins known toenhance the immune response (e.g. chemokines), to said antigens andimmunogens.

Cardiovascular Disorders

Polynucleotides or polypeptides, or agonists or antagonists of theinvention may be used to treat, prevent, and/or diagnose cardiovasculardiseases, disorders, and/or conditions, including peripheral arterydisease, such as limb ischemia.

Cardiovascular diseases, disorders, and/or conditions includecardiovascular abnormalities, such as arterio-arterial fistula,arteriovenous fistula, cerebral arteriovenous malformations, congenitalheart defects, pulmonary atresia, and Scimitar Syndrome. Congenitalheart defects include aortic coarctation, cor triatriatum, coronaryvessel anomalies, crisscross heart, dextrocardia, patent ductusarteriosus, Ebstein's anomaly, Eisenmenger complex, hypoplastic leftheart syndrome, levocardia, tetralogy of fallot, transposition of greatvessels, double outlet right ventricle, tricuspid atresia, persistenttruncus arteriosus, and heart septal defects, such as aortopulmonaryseptal defect, endocardial cushion defects, Lutembacher's Syndrome,trilogy of Fallot, ventricular heart septal defects.

Cardiovascular diseases, disorders, and/or conditions also include heartdisease, such as arrhythmias, carcinoid heart disease, high cardiacoutput, low cardiac output, cardiac tamponade, endocarditis (includingbacterial), heart aneurysm, cardiac arrest, congestive heart failure,congestive cardiomyopathy, paroxysmal dyspnea, cardiac edema, hearthypertrophy, congestive cardiomyopathy, left ventricular hypertrophy,right ventricular hypertrophy, post-infarction heart rupture,ventricular septal rupture, heart valve diseases, myocardial diseases,myocardial ischemia, pericardial effusion, pericarditis (includingconstrictive and tuberculous), pneumopericardium, postpericardiotomysyndrome, pulmonary heart disease, rheumatic heart disease, ventriculardysfunction, hyperemia, cardiovascular pregnancy complications, ScimitarSyndrome, cardiovascular syphilis, and cardiovascular tuberculosis.

Arrhythmias include sinus arrhythmia, atrial fibrillation, atrialflutter, bradycardia, extrasystole, Adams-Stokes Syndrome, bundle-branchblock, sinoatrial block, long QT syndrome, parasystole,Lown-Ganong-Levine Syndrome, Mahaim-type pre-excitation syndrome,Wolff-Parkinson-White syndrome, sick sinus syndrome, tachycardias, andventricular fibrillation. Tachycardias include paroxysmal tachycardia,supraventricular tachycardia, accelerated idioventricular rhythm,atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia,ectopic junctional tachycardia, sinoatrial nodal reentry tachycardia,sinus tachycardia, Torsades de Pointes, and ventricular tachycardia.

Heart valve disease include aortic valve insufficiency, aortic valvestenosis, hear murmurs, aortic valve prolapse, mitral valve prolapse,tricuspid valve prolapse, mitral valve insufficiency, mitral valvestenosis, pulmonary atresia, pulmonary valve insufficiency, pulmonaryvalve stenosis, tricuspid atresia, tricuspid valve insufficiency, andtricuspid valve stenosis.

Myocardial diseases include alcoholic cardiomyopathy, congestivecardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvularstenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy,Chagas cardiomyopathy, endocardial fibroelastosis, endomyocardialfibrosis, Kearns Syndrome, myocardial reperfusion injury, andmyocarditis.

Myocardial ischemias include coronary disease, such as angina pectoris,coronary aneurysm, coronary arteriosclerosis, coronary thrombosis,coronary vasospasm, myocardial infarction and myocardial stunning.

Cardiovascular diseases also include vascular diseases such asaneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis,Hippel-Lindau Disease, Klippel-Trenaunay-Weber Syndrome, Sturge-WeberSyndrome, angioneurotic edema, aortic diseases, Takayasu's Arteritis,aortitis, Leriche's Syndrome, arterial occlusive diseases, arteritis,enarteritis, polyarteritis nodosa, cerebrovascular diseases, disorders,and/or conditions, diabetic angiopathies, diabetic retinopathy,embolisms, thrombosis, erythromelalgia, hemorrhoids, hepaticveno-occlusive disease, hypertension, hypotension, ischemia, peripheralvascular diseases, phlebitis, pulmonary veno-occlusive disease,Raynaud's disease, CREST syndrome, retinal vein occlusion, Scimitarsyndrome, superior vena cava syndrome, telangiectasia, ataciatelangiectasia, hereditary hemorrhagic telangiectasia, varicocele,varicose veins, varicose ulcer, vasculitis, and venous insufficiency.

Aneurysms include dissecting aneurysms, false aneurysms, infectedaneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms,coronary aneurysms, heart aneurysms, and iliac aneurysms.

Arterial occlusive diseases include arteriosclerosis, intermittentclaudication, carotid stenosis, fibromuscular dysplasias, mesentericvascular occlusion, Moyamoya disease, renal artery obstruction, retinalartery occlusion, and thromboangiitis obliterans.

Cerebrovascular diseases, disorders, and/or conditions include carotidartery diseases, cerebral amyloid angiopathy, cerebral aneurysm,cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenousmalformation, cerebral artery diseases, cerebral embolism andthrombosis, carotid artery thrombosis, sinus thrombosis, Wallenberg'ssyndrome, cerebral hemorrhage, epidural hematoma, subdural hematoma,subaraxhnoid hemorrhage, cerebral infarction, cerebral ischemia(including transient), subclavian steal syndrome, periventricularleukomalacia, vascular headache, cluster headache, migraine, andvertebrobasilar insufficiency.

Embolisms include air embolisms, amniotic fluid embolisms, cholesterolembolisms, blue toe syndrome, fat embolisms, pulmonary embolisms, andthromoboembolisms. Thrombosis include coronary thrombosis, hepatic veinthrombosis, retinal vein occlusion, carotid artery thrombosis, sinusthrombosis, Wallenberg's syndrome, and thrombophlebitis.

Ischemia includes cerebral ischemia, ischemic colitis, compartmentsyndromes, anterior compartment syndrome, myocardial ischemia,reperfusion injuries, and peripheral limb ischemia. Vasculitis includesaortitis, arteritis, Behcet's Syndrome, Churg-Strauss Syndrome,mucocutaneous lymph node syndrome, thromboangiitis obliterans,hypersensitivity vasculitis, Schoenlein-Henoch purpura, allergiccutaneous vasculitis, and Wegener's granulomatosis.

Polynucleotides or polypeptides, or agonists or antagonists of theinvention, are especially effective for the treatment of critical limbischemia and coronary disease.

Polypeptides may be administered using any method known in the art,including, but not limited to, direct needle injection at the deliverysite, intravenous injection, topical administration, catheter infusion,biolistic injectors, particle accelerators, gelfoam sponge depots, othercommercially available depot materials, osmotic pumps, oral orsuppositorial solid pharmaceutical formulations, decanting or topicalapplications during surgery, aerosol delivery. Such methods are known inthe art. Polypeptides of the invention may be administered as part of aTherapeutic, described in more detail below. Methods of deliveringpolynucleotides of the invention are described in more detail herein.

Anti-Angiogenesis Activity

The naturally occurring balance between endogenous stimulators andinhibitors of angiogenesis is one in which inhibitory influencespredominate. Rastinejad et al., Cell 56:345-355 (1989). In those rareinstances in which neovascularization occurs under normal physiologicalconditions, such as wound healing, organ regeneration, embryonicdevelopment, and female reproductive processes, angiogenesis isstringently regulated and spatially and temporally delimited. Underconditions of pathological angiogenesis such as that characterizingsolid tumor growth, these regulatory controls fail. Unregulatedangiogenesis becomes pathologic and sustains progression of manyneoplastic and non-neoplastic diseases. A number of serious diseases aredominated by abnormal neovascularization including solid tumor growthand metastases, arthritis, some types of eye diseases, disorders, and/orconditions, and psoriasis. See, e.g., reviews by Moses et al., Biotech.9:630-634 (1991); Folkman et al., N. Engl. J. Med., 333:1757-1763(1995); Auerbach et al., J. Microvasc. Res. 29:401-411 (1985); Folkman,Advances in Cancer Research, eds. Klein and Weinhouse, Academic Press,New York, pp. 175-203 (1985); Patz, Am. J. Opthalmol. 94:715-743 (1982);and Folkman et al., Science 221:719-725 (1983). In a number ofpathological conditions, the process of angiogenesis contributes to thedisease state. For example, significant data have accumulated whichsuggest that the growth of solid tumors is dependent on angiogenesis.Folkman and Klagsbrun, Science 235:442-447 (1987).

The present invention provides for treatment of diseases, disorders,and/or conditions associated with neovascularization by administrationof the polynucleotides and/or polypeptides of the invention, as well asagonists or antagonists of the present invention. Malignant andmetastatic conditions which can be treated with the polynucleotides andpolypeptides, or agonists or antagonists of the invention include, butare not limited to, malignancies, solid tumors, and cancers describedherein and otherwise known in the art (for a review of such disorders,see Fishman et al., Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia(1985)). Thus, the present invention provides a method of treating,preventing, and/or diagnosing an angiogenesis-related disease and/ordisorder, comprising administering to an individual in need thereof atherapeutically effective amount of a polynucleotide, polypeptide,antagonist and/or agonist of the invention. For example,polynucleotides, polypeptides, antagonists and/or agonists may beutilized in a variety of additional methods in order to therapeuticallytreat or prevent a cancer or tumor. Cancers which may be treated,prevented, and/or diagnosed with polynucleotides, polypeptides,antagonists and/or agonists include, but are not limited to solidtumors, including prostate, lung, breast, ovarian, stomach, pancreas,larynx, esophagus, testes, liver, parotid, biliary tract, colon, rectum,cervix, uterus, endometrium, kidney, bladder, thyroid cancer; primarytumors and metastases; melanomas; glioblastoma; Kaposi's sarcoma;leiomyosarcoma; non-small cell lung cancer; colorectal cancer; advancedmalignancies; and blood born tumors such as leukemias. For example,polynucleotides, polypeptides, antagonists and/or agonists may bedelivered topically, in order to treat or prevent cancers such as skincancer, head and neck tumors, breast tumors, and Kaposi's sarcoma.

Within yet other aspects, polynucleotides, polypeptides, antagonistsand/or agonists may be utilized to treat superficial forms of bladdercancer by, for example, intravesical administration. Polynucleotides,polypeptides, antagonists and/or agonists may be delivered directly intothe tumor, or near the tumor site, via injection or a catheter. Ofcourse, as the artisan of ordinary skill will appreciate, theappropriate mode of administration will vary according to the cancer tobe treated. Other modes of delivery are discussed herein.

Polynucleotides, polypeptides, antagonists and/or agonists may be usefulin treating, preventing, and/or diagnosing other diseases, disorders,and/or conditions, besides cancers, which involve angiogenesis. Thesediseases, disorders, and/or conditions include, but are not limited to:benign tumors, for example hemangiomas, acoustic neuromas,neurofibromas, trachomas, and pyogenic granulomas; artherosclericplaques; ocular angiogenic diseases, for example, diabetic retinopathy,retinopathy of prematurity, macular degeneration, corneal graftrejection, neovascular glaucoma, retrolental fibroplasia, rubeosis,retinoblastoma, uvietis and Pterygia (abnormal blood vessel growth) ofthe eye; rheumatoid arthritis; psoriasis; delayed wound healing;endometriosis; vasculogenesis; granulations; hypertrophic scars(keloids); nonunion fractures; scleroderma; trachoma; vascularadhesions; myocardial angiogenesis; coronary collaterals; cerebralcollaterals; arteriovenous malformations; ischemic limb angiogenesis;Osler-Webber Syndrome; plaque neovascularization; telangiectasia;hemophiliac joints; angiofibroma; fibromuscular dysplasia; woundgranulation; Crohn's disease; and atherosclerosis.

For example, within one aspect of the present invention methods areprovided for treating, preventing, and/or diagnosing hypertrophic scarsand keloids, comprising the step of administering a polynucleotide,polypeptide, antagonist and/or agonist of the invention to ahypertrophic scar or keloid.

Within one embodiment of the present invention polynucleotides,polypeptides, antagonists and/or agonists are directly injected into ahypertrophic scar or keloid, in order to prevent the progression ofthese lesions. This therapy is of particular value in the prophylactictreatment of conditions which are known to result in the development ofhypertrophic scars and keloids (e.g., burns), and is preferablyinitiated after the proliferative phase has had time to progress(approximately 14 days after the initial injury), but beforehypertrophic scar or keloid development. As noted above, the presentinvention also provides methods for treating, preventing, and/ordiagnosing neovascular diseases of the eye, including for example,corneal neovascularization, neovascular glaucoma, proliferative diabeticretinopathy, retrolental fibroplasia and macular degeneration.

Moreover, Ocular diseases, disorders, and/or conditions associated withneovascularization which can be treated, prevented, and/or diagnosedwith the polynucleotides and polypeptides of the present invention(including agonists and/or antagonists) include, but are not limited to:neovascular glaucoma, diabetic retinopathy, retinoblastoma, retrolentalfibroplasia, uveitis, retinopathy of prematurity macular degeneration,corneal graft neovascularization, as well as other eye inflammatorydiseases, ocular tumors and diseases associated with choroidal or irisneovascularization. See, e.g., reviews by Waltman et al., Am. J.Ophthal. 85:704-710 (1978) and Gartner et al., Surv. Ophthal. 22:291-312(1978).

Thus, within one aspect of the present invention methods are providedfor treating or preventing neovascular diseases of the eye such ascorneal neovascularization (including corneal graft neovascularization),comprising the step of administering to a patient a therapeuticallyeffective amount of a compound (as described above) to the cornea, suchthat the formation of blood vessels is inhibited. Briefly, the cornea isa tissue which normally lacks blood vessels. In certain pathologicalconditions however, capillaries may extend into the cornea from thepericorneal vascular plexus of the limbus. When the cornea becomesvascularized, it also becomes clouded, resulting in a decline in thepatient's visual acuity. Visual loss may become complete if the corneacompletely opacitates. A wide variety of diseases, disorders, and/orconditions can result in corneal neovascularization, including forexample, corneal infections (e.g., trachoma, herpes simplex keratitis,leishmaniasis and onchocerciasis), immunological processes (e.g., graftrejection and Stevens-Johnson's syndrome), alkali burns, trauma,inflammation (of any cause), toxic and nutritional deficiency states,and as a complication of wearing contact lenses.

Within particularly preferred embodiments of the invention, may beprepared for topical administration in saline (combined with any of thepreservatives and antimicrobial agents commonly used in ocularpreparations), and administered in eyedrop form. The solution orsuspension may be prepared in its pure form and administered severaltimes daily. Alternatively, anti-angiogenic compositions, prepared asdescribed above, may also be administered directly to the cornea. Withinpreferred embodiments, the anti-angiogenic composition is prepared witha muco-adhesive polymer which binds to cornea. Within furtherembodiments, the anti-angiogenic factors or anti-angiogenic compositionsmay be utilized as an adjunct to conventional steroid therapy. Topicaltherapy may also be useful prophylactically in corneal lesions which areknown to have a high probability of inducing an angiogenic response(such as chemical burns). In these instances the treatment, likely incombination with steroids, may be instituted immediately to help preventsubsequent complications.

Within other embodiments, the compounds described above may be injecteddirectly into the corneal stroma by an ophthalmologist under microscopicguidance. The preferred site of injection may vary with the morphologyof the individual lesion, but the goal of the administration would be toplace the composition at the advancing front of the vasculature (i.e.,interspersed between the blood vessels and the normal cornea). In mostcases this would involve perilimbic corneal injection to “protect” thecornea from the advancing blood vessels. This method may also beutilized shortly after a corneal insult in order to prophylacticallyprevent corneal neovascularization. In this situation the material couldbe injected in the perilimbic cornea interspersed between the corneallesion and its undesired potential limbic blood supply. Such methods mayalso be utilized in a similar fashion to prevent capillary invasion oftransplanted corneas. In a sustained-release form injections might onlybe required 2-3 times per year. A steroid could also be added to theinjection solution to reduce inflammation resulting from the injectionitself.

Within another aspect of the present invention, methods are provided fortreating or preventing neovascular glaucoma, comprising the step ofadministering to a patient a therapeutically effective amount of apolynucleotide, polypeptide, antagonist and/or agonist to the eye, suchthat the formation of blood vessels is inhibited. In one embodiment, thecompound may be administered topically to the eye in order to treat orprevent early forms of neovascular glaucoma. Within other embodiments,the compound may be implanted by injection into the region of theanterior chamber angle. Within other embodiments, the compound may alsobe placed in any location such that the compound is continuouslyreleased into the aqueous humor. Within another aspect of the presentinvention, methods are provided for treating or preventing proliferativediabetic retinopathy, comprising the step of administering to a patienta therapeutically effective amount of a polynucleotide, polypeptide,antagonist and/or agonist to the eyes, such that the formation of bloodvessels is inhibited.

Within particularly preferred embodiments of the invention,proliferative diabetic retinopathy may be treated by injection into theaqueous humor or the vitreous, in order to increase the localconcentration of the polynucleotide, polypeptide, antagonist and/oragonist in the retina. Preferably, this treatment should be initiatedprior to the acquisition of severe disease requiring photocoagulation.

Within another aspect of the present invention, methods are provided fortreating or preventing retrolental fibroplasia, comprising the step ofadministering to a patient a therapeutically effective amount of apolynucleotide, polypeptide, antagonist and/or agonist to the eye, suchthat the formation of blood vessels is inhibited. The compound may beadministered topically, via intravitreous injection and/or viaintraocular implants.

Additionally, diseases, disorders, and/or conditions which can betreated, prevented, and/or diagnosed with the polynucleotides,polypeptides, agonists and/or agonists include, but are not limited to,hemangioma, arthritis, psoriasis, angiofibroma, atherosclerotic plaques,delayed wound healing, granulations, hemophilic joints, hypertrophicscars, nonunion fractures, Osler-Weber syndrome, pyogenic granuloma,scleroderma, trachoma, and vascular adhesions.

Moreover, diseases, disorders, and/or conditions and/or states, whichcan be treated, prevented, and/or diagnosed with the polynucleotides,polypeptides, agonists and/or agonists include, but are not limited to,solid tumors, blood born tumors such as leukemias, tumor metastasis,Kaposi's sarcoma, benign tumors, for example hemangiomas, acousticneuromas, neurofibromas, trachomas, and pyogenic granulomas, rheumatoidarthritis, psoriasis, ocular angiogenic diseases, for example, diabeticretinopathy, retinopathy of prematurity, macular degeneration, cornealgraft rejection, neovascular glaucoma, retrolental fibroplasia,rubeosis, retinoblastoma, and uvietis, delayed wound healing,endometriosis, vascluogenesis, granulations, hypertrophic scars(keloids), nonunion fractures, scleroderma, trachoma, vascularadhesions, myocardial angiogenesis, coronary collaterals, cerebralcollaterals, arteriovenous malformations, ischemic limb angiogenesis,Osler-Webber Syndrome, plaque neovascularization, telangiectasia,hemophiliac joints, angiofibroma fibromuscular dysplasia, woundgranulation, Crohn's disease, atherosclerosis, birth control agent bypreventing vascularization required for embryo implantation controllingmenstruation, diseases that have angiogenesis as a pathologicconsequence such as cat scratch disease (Rochele minalia quintosa),ulcers (Helicobacter pylori), Bartonellosis and bacillary angiomatosis.

In one aspect of the birth control method, an amount of the compoundsufficient to block embryo implantation is administered before or afterintercourse and fertilization have occurred, thus providing an effectivemethod of birth control, possibly a “morning after” method.Polynucleotides, polypeptides, agonists and/or agonists may also be usedin controlling menstruation or administered as either a peritoneallavage fluid or for peritoneal implantation in the treatment ofendometriosis.

Polynucleotides, polypeptides, agonists and/or agonists of the presentinvention may be incorporated into surgical sutures in order to preventstitch granulomas.

Polynucleotides, polypeptides, agonists and/or agonists may be utilizedin a wide variety of surgical procedures. For example, within one aspectof the present invention a compositions (in the form of, for example, aspray or film) may be utilized to coat or spray an area prior to removalof a tumor, in order to isolate normal surrounding tissues frommalignant tissue, and/or to prevent the spread of disease to surroundingtissues. Within other aspects of the present invention, compositions(e.g., in the form of a spray) may be delivered via endoscopicprocedures in order to coat tumors, or inhibit angiogenesis in a desiredlocale. Within yet other aspects of the present invention, surgicalmeshes which have been coated with anti-angiogenic compositions of thepresent invention may be utilized in any procedure wherein a surgicalmesh might be utilized. For example, within one embodiment of theinvention a surgical mesh laden with an anti-angiogenic composition maybe utilized during abdominal cancer resection surgery (e.g., subsequentto colon resection) in order to provide support to the structure, and torelease an amount of the anti-angiogenic factor.

Within further aspects of the present invention, methods are providedfor treating tumor excision sites, comprising administering apolynucleotide, polypeptide, agonist and/or agonist to the resectionmargins of a tumor subsequent to excision, such that the localrecurrence of cancer and the formation of new blood vessels at the siteis inhibited. Within one embodiment of the invention, theanti-angiogenic compound is administered directly to the tumor excisionsite (e.g., applied by swabbing, brushing or otherwise coating theresection margins of the tumor with the anti-angiogenic compound).Alternatively, the anti-angiogenic compounds may be incorporated intoknown surgical pastes prior to administration. Within particularlypreferred embodiments of the invention, the anti-angiogenic compoundsare applied after hepatic resections for malignancy, and afterneurosurgical operations.

Within one aspect of the present invention, polynucleotides,polypeptides, agonists and/or agonists may be administered to theresection margin of a wide variety of tumors, including for example,breast, colon, brain and hepatic tumors. For example, within oneembodiment of the invention, anti-angiogenic compounds may beadministered to the site of a neurological tumor subsequent to excision,such that the formation of new blood vessels at the site are inhibited.

The polynucleotides, polypeptides, agonists and/or agonists of thepresent invention may also be administered along with otheranti-angiogenic factors. Representative examples of otheranti-angiogenic factors include: Anti-Invasive Factor, retinoic acid andderivatives thereof, paclitaxel, Suramin, Tissue Inhibitor ofMetalloproteinase-1, Tissue Inhibitor of Metalloproteinase-2,Plasminogen Activator Inhibitor-1, Plasminogen Activator Inhibitor-2,and various forms of the lighter “d group” transition metals.

Lighter “d group” transition metals include, for example, vanadium,molybdenum, tungsten, titanium, niobium, and tantalum species. Suchtransition metal species may form transition metal complexes. Suitablecomplexes of the above-mentioned transition metal species include oxotransition metal complexes.

Representative examples of vanadium complexes include oxo vanadiumcomplexes such as vanadate and vanadyl complexes. Suitable vanadatecomplexes include metavanadate and orthovanadate complexes such as, forexample, ammonium metavanadate, sodium metavanadate, and sodiumorthovanadate. Suitable vanadyl complexes include, for example, vanadylacetylacetonate and vanadyl sulfate including vanadyl sulfate hydratessuch as vanadyl sulfate mono- and trihydrates.

Representative examples of tungsten and molybdenum complexes alsoinclude oxo complexes. Suitable oxo tungsten complexes include tungstateand tungsten oxide complexes. Suitable tungstate complexes includeammonium tungstate, calcium tungstate, sodium tungstate dihydrate, andtungstic acid. Suitable tungsten oxides include tungsten (IV) oxide andtungsten (VI) oxide. Suitable oxo molybdenum complexes includemolybdate, molybdenum oxide, and molybdenyl complexes. Suitablemolybdate complexes include ammonium molybdate and its hydrates, sodiummolybdate and its hydrates, and potassium molybdate and its hydrates.Suitable molybdenum oxides include molybdenum (VI) oxide, molybdenum(VI) oxide, and molybdic acid. Suitable molybdenyl complexes include,for example, molybdenyl acetylacetonate. Other suitable tungsten andmolybdenum complexes include hydroxo derivatives derived from, forexample, glycerol, tartaric acid, and sugars.

A wide variety of other anti-angiogenic factors may also be utilizedwithin the context of the present invention. Representative examplesinclude platelet factor 4; protamine sulphate; sulphated chitinderivatives (prepared from queen crab shells), (Murata et al., CancerRes. 51:22-26, 1991); Sulphated Polysaccharide Peptidoglycan Complex(SP-PG) (the function of this compound may be enhanced by the presenceof steroids such as estrogen, and tamoxifen citrate); Staurosporine;modulators of matrix metabolism, including for example, proline analogs,cishydroxyproline, d,L-3,4-dehydroproline, Thiaproline,alpha,alpha-dipyridyl, aminopropionitrile fumarate;4-propyl-5-(4-pyridinyl)-2 (3H)-oxazolone; Methotrexate; Mitoxantrone;Heparin; Interferons; 2 Macroglobulin-serum; ChIMP-3 (Pavloff et al., J.Bio. Chem. 267:17321-17326, 1992); Chymostatin (Tomkinson et al.,Biochem J. 286:475-480, 1992); Cyclodextrin Tetradecasulfate;Eponemycin; Camptothecin; Fumagillin (Ingber et al., Nature 348:555-557,1990); Gold Sodium Thiomalate (“GST”; Matsubara and Ziff, J. Clin.Invest. 79:1440-1446, 1987); anticollagenase-serum; alpha2-antiplasmin(Holmes et al., J. Biol. Chem. 262(4):1659-1664, 1987); Bisantrene(National Cancer Institute); Lobenzarit disodium(N-(2)-carboxyphenyl-4-chloroanthronilic acid disodium or “CCA”;Takeuchi et al., Agents Actions 36:312-316, 1992); Thalidomide;Angostatic steroid; AGM-1470; carboxynaminolmidazole; andmetalloproteinase inhibitors such as BB94.

Diseases at the Cellular Level

Diseases associated with increased cell survival or the inhibition ofapoptosis that could be treated, prevented, and/or diagnosed by thepolynucleotides or polypeptides and/or antagonists or agonists of theinvention, include cancers (such as follicular lymphomas, carcinomaswith p53 mutations, and hormone-dependent tumors, including, but notlimited to colon cancer, cardiac tumors, pancreatic cancer, melanoma,retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicularcancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma,endothelioma, osteoblastoma, osteoclastoma, osteosarcoma,chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi'ssarcoma and ovarian cancer); autoimmune diseases, disorders, and/orconditions (such as, multiple sclerosis, Sjogren's syndrome, Hashimoto'sthyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease,polymyositis, systemic lupus erythematosus and immune-relatedglomerulonephritis and rheumatoid arthritis) and viral infections (suchas herpes viruses, pox viruses and adenoviruses), inflammation, graft v.host disease, acute graft rejection, and chronic graft rejection. Inpreferred embodiments, the polynucleotides or polypeptides, and/oragonists or antagonists of the invention are used to inhibit growth,progression, and/or metastasis of cancers, in particular those listedabove.

Additional diseases or conditions associated with increased cellsurvival that could be treated, prevented or diagnosed by thepolynucleotides or polypeptides, or agonists or antagonists of theinvention, include, but are not limited to, progression, and/ormetastases of malignancies and related disorders such as leukemia(including acute leukemias (e.g., acute lymphocytic leukemia, acutemyelocytic leukemia (including myeloblastic, promyelocytic,myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias(e.g., chronic myelocytic (granulocytic) leukemia and chroniclymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin'sdisease and non-Hodgkin's disease), multiple myeloma, Waldenstrom'smacroglobulinemia, heavy chain disease, and solid tumors including, butnot limited to, sarcomas and carcinomas such as fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, andretinoblastoma.

Diseases associated with increased apoptosis that could be treated,prevented, and/or diagnosed by the polynucleotides or polypeptides,and/or agonists or antagonists of the invention, include AIDS;neurodegenerative diseases, disorders, and/or conditions (such asAlzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis,Retinitis pigmentosa, Cerebellar degeneration and brain tumor or priorassociated disease); autoimmune diseases, disorders, and/or conditions(such as, multiple sclerosis, Sjogren's syndrome, Hashimoto'sthyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease,polymyositis, systemic lupus erythematosus and immune-relatedglomerulonephritis and rheumatoid arthritis) myelodysplastic syndromes(such as aplastic anemia), graft v. host disease, ischemic injury (suchas that caused by myocardial infarction, stroke and reperfusion injury),liver injury (e.g., hepatitis related liver injury, ischemia/reperfusioninjury, cholestosis (bile duct injury) and liver cancer); toxin-inducedliver disease (such as that caused by alcohol), septic shock, cachexiaand anorexia.

Wound Healing and Epithelial Cell Proliferation

In accordance with yet a further aspect of the present invention, thereis provided a process for utilizing the polynucleotides or polypeptides,and/or agonists or antagonists of the invention, for therapeuticpurposes, for example, to stimulate epithelial cell proliferation andbasal keratinocytes for the purpose of wound healing, and to stimulatehair follicle production and healing of dermal wounds. Polynucleotidesor polypeptides, as well as agonists or antagonists of the invention,may be clinically useful in stimulating wound healing including surgicalwounds, excisional wounds, deep wounds involving damage of the dermisand epidermis, eye tissue wounds, dental tissue wounds, oral cavitywounds, diabetic ulcers, dermal ulcers, cubitus ulcers, arterial ulcers,venous stasis ulcers, burns resulting from heat exposure or chemicals,and other abnormal wound healing conditions such as uremia,malnutrition, vitamin deficiencies and complications associated withsystemic treatment with steroids, radiation therapy and antineoplasticdrugs and antimetabolites. Polynucleotides or polypeptides, and/oragonists or antagonists of the invention, could be used to promotedermal reestablishment subsequent to dermal loss

The polynucleotides or polypeptides, and/or agonists or antagonists ofthe invention, could be used to increase the adherence of skin grafts toa wound bed and to stimulate re-epithelialization from the wound bed.The following are a non-exhaustive list of grafts that polynucleotidesor polypeptides, agonists or antagonists of the invention, could be usedto increase adherence to a wound bed: autografts, artificial skin,allografts, autodermic graft, autoepidermic grafts, avacular grafts,Blair-Brown grafts, bone graft, brephoplastic grafts, cutis graft,delayed graft, dermic graft, epidermic graft, fascia graft, fullthickness graft, heterologous graft, xenograft, homologous graft,hyperplastic graft, lamellar graft, mesh graft, mucosal graft,Ollier-Thiersch graft, omenpal graft, patch graft, pedicle graft,penetrating graft, split skin graft, thick split graft. Thepolynucleotides or polypeptides, and/or agonists or antagonists of theinvention, can be used to promote skin strength and to improve theappearance of aged skin.

It is believed that the polynucleotides or polypeptides, and/or agonistsor antagonists of the invention, will also produce changes in hepatocyteproliferation, and epithelial cell proliferation in the lung, breast,pancreas, stomach, small intestine, and large intestine. Thepolynucleotides or polypeptides, and/or agonists or antagonists of theinvention, could promote proliferation of epithelial cells such assebocytes, hair follicles, hepatocytes, type II pneumocytes,mucin-producing goblet cells, and other epithelial cells and theirprogenitors contained within the skin, lung, liver, and gastrointestinaltract. The polynucleotides or polypeptides, and/or agonists orantagonists of the invention, may promote proliferation of endothelialcells, keratinocytes, and basal keratinocytes.

The polynucleotides or polypeptides, and/or agonists or antagonists ofthe invention, could also be used to reduce the side effects of guttoxicity that result from radiation, chemotherapy treatments or viralinfections. The polynucleotides or polypeptides, and/or agonists orantagonists of the invention, may have a cytoprotective effect on thesmall intestine mucosa. The polynucleotides or polypeptides, and/oragonists or antagonists of the invention, may also stimulate healing ofmucositis (mouth ulcers) that result from chemotherapy and viralinfections.

The polynucleotides or polypeptides, and/or agonists or antagonists ofthe invention, could further be used in full regeneration of skin infull and partial thickness skin defects, including burns, (i.e.,repopulation of hair follicles, sweat glands, and sebaceous glands),treatment of other skin defects such as psoriasis. The polynucleotidesor polypeptides, and/or agonists or antagonists of the invention, couldbe used to treat epidermolysis bullosa, a defect in adherence of theepidermis to the underlying dermis which results in frequent, open andpainful blisters by accelerating reepithelialization of these lesions.The polynucleotides or polypeptides, and/or agonists or antagonists ofthe invention, could also be used to treat gastric and doudenal ulcersand help heal by scar formation of the mucosal lining and regenerationof glandular mucosa and duodenal mucosal lining more rapidly.Inflamamatory bowel diseases, such as Crohn's disease and ulcerativecolitis, are diseases which result in destruction of the mucosal surfaceof the small or large intestine, respectively. Thus, the polynucleotidesor polypeptides, and/or agonists or antagonists of the invention, couldbe used to promote the resurfacing of the mucosal surface to aid morerapid healing and to prevent progression of inflammatory bowel disease.Treatment with the polynucleotides or polypeptides, and/or agonists orantagonists of the invention, is expected to have a significant effecton the production of mucus throughout the gastrointestinal tract andcould be used to protect the intestinal mucosa from injurious substancesthat are ingested or following surgery. The polynucleotides orpolypeptides, and/or agonists or antagonists of the invention, could beused to treat diseases associate with the under expression of thepolynucleotides of the invention.

Moreover, the polynucleotides or polypeptides, and/or agonists orantagonists of the invention, could be used to prevent and heal damageto the lungs due to various pathological states. A growth factor such asthe polynucleotides or polypeptides, and/or agonists or antagonists ofthe invention, which could stimulate proliferation and differentiationand promote the repair of alveoli and brochiolar epithelium to preventor treat acute or chronic lung damage. For example, emphysema, whichresults in the progressive loss of aveoli, and inhalation injuries,i.e., resulting from smoke inhalation and burns, that cause necrosis ofthe bronchiolar epithelium and alveoli could be effectively treated,prevented, and/or diagnosed using the polynucleotides or polypeptides,and/or agonists or antagonists of the invention. Also, thepolynucleotides or polypeptides, and/or agonists or antagonists of theinvention, could be used to stimulate the proliferation of anddifferentiation of type II pneumocytes, which may help treat or preventdisease such as hyaline membrane diseases, such as infant respiratorydistress syndrome and bronchopulmonary displasia, in premature infants.

The polynucleotides or polypeptides, and/or agonists or antagonists ofthe invention, could stimulate the proliferation and differentiation ofhepatocytes and, thus, could be used to alleviate or treat liverdiseases and pathologies such as fulminant liver failure caused bycirrhosis, liver damage caused by viral hepatitis and toxic substances(i.e., acetaminophen, carbon tetraholoride and other hepatotoxins knownin the art).

In addition, the polynucleotides or polypeptides, and/or agonists orantagonists of the invention, could be used treat or prevent the onsetof diabetes mellitus. In patients with newly diagnosed Types I and IIdiabetes, where some islet cell function remains, the polynucleotides orpolypeptides, and/or agonists or antagonists of the invention, could beused to maintain the islet function so as to alleviate, delay or preventpermanent manifestation of the disease. Also, the polynucleotides orpolypeptides, and/or agonists or antagonists of the invention, could beused as an auxiliary in islet cell transplantation to improve or promoteislet cell function.

Neurological Diseases

Nervous system diseases, disorders, and/or conditions, which can betreated, prevented, and/or diagnosed with the compositions of theinvention (e.g., polypeptides, polynucleotides, and/or agonists orantagonists), include, but are not limited to, nervous system injuries,and diseases, disorders, and/or conditions which result in either adisconnection of axons, a diminution or degeneration of neurons, ordemyelination. Nervous system lesions which may be treated, prevented,and/or diagnosed in a patient (including human and non-human mammalianpatients) according to the invention, include but are not limited to,the following lesions of either the central (including spinal cord,brain) or peripheral nervous systems: (1) ischemic lesions, in which alack of oxygen in a portion of the nervous system results in neuronalinjury or death, including cerebral infarction or ischemia, or spinalcord infarction or ischemia; (2) traumatic lesions, including lesionscaused by physical injury or associated with surgery, for example,lesions which sever a portion of the nervous system, or compressioninjuries; (3) malignant lesions, in which a portion of the nervoussystem is destroyed or injured by malignant tissue which is either anervous system associated malignancy or a malignancy derived fromnon-nervous system tissue; (4) infectious lesions, in which a portion ofthe nervous system is destroyed or injured as a result of infection, forexample, by an abscess or associated with infection by humanimmunodeficiency virus, herpes zoster, or herpes simplex virus or withLyme disease, tuberculosis, syphilis; (5) degenerative lesions, in whicha portion of the nervous system is destroyed or injured as a result of adegenerative process including but not limited to degenerationassociated with Parkinson's disease, Alzheimer's disease, Huntington'schorea, or amyotrophic lateral sclerosis (ALS); (6) lesions associatedwith nutritional diseases, disorders, and/or conditions, in which aportion of the nervous system is destroyed or injured by a nutritionaldisorder or disorder of metabolism including but not limited to, vitaminB12 deficiency, folic acid deficiency, Wernicke disease, tobacco-alcoholamblyopia, Marchiafava-Bignami disease (primary degeneration of thecorpus callosum), and alcoholic cerebellar degeneration; (7)neurological lesions associated with systemic diseases including, butnot limited to, diabetes (diabetic neuropathy, Bell's palsy), systemiclupus erythematosus, carcinoma, or sarcoidosis; (8) lesions caused bytoxic substances including alcohol, lead, or particular neurotoxins; and(9) demyelinated lesions in which a portion of the nervous system isdestroyed or injured by a demyelinating disease including, but notlimited to, multiple sclerosis, human immunodeficiency virus-associatedmyelopathy, transverse myelopathy or various etiologies, progressivemultifocal leukoencephalopathy, and central pontine myelinolysis.

In a preferred embodiment, the polypeptides, polynucleotides, oragonists or antagonists of the invention are used to protect neuralcells from the damaging effects of cerebral hypoxia. According to thisembodiment, the compositions of the invention are used to treat,prevent, and/or diagnose neural cell injury associated with cerebralhypoxia. In one aspect of this embodiment, the polypeptides,polynucleotides, or agonists or antagonists of the invention are used totreat, prevent, and/or diagnose neural cell injury associated withcerebral ischemia. In another aspect of this embodiment, thepolypeptides, polynucleotides, or agonists or antagonists of theinvention are used to treat, prevent, and/or diagnose neural cell injuryassociated with cerebral infarction. In another aspect of thisembodiment, the polypeptides, polynucleotides, or agonists orantagonists of the invention are used to treat, prevent, and/or diagnoseor prevent neural cell injury associated with a stroke. In a furtheraspect of this embodiment, the polypeptides, polynucleotides, oragonists or antagonists of the invention are used to treat, prevent,and/or diagnose neural cell injury associated with a heart attack.

The compositions of the invention which are useful for treating orpreventing a nervous system disorder may be selected by testing forbiological activity in promoting the survival or differentiation ofneurons. For example, and not by way of limitation, compositions of theinvention which elicit any of the following effects may be usefulaccording to the invention: (1) increased survival time of neurons inculture; (2) increased sprouting of neurons in culture or in vivo; (3)increased production of a neuron-associated molecule in culture or invivo, e.g., choline acetyltransferase or acetylcholinesterase withrespect to motor neurons; or (4) decreased symptoms of neurondysfunction in vivo. Such effects may be measured by any method known inthe art. In preferred, non-limiting embodiments, increased survival ofneurons may routinely be measured using a method set forth herein orotherwise known in the art, such as, for example, the method set forthin Arakawa et al. (J. Neurosci. 10:3507-3515 (1990)); increasedsprouting of neurons may be detected by methods known in the art, suchas, for example, the methods set forth in Pestronk et al. (Exp. Neurol.70:65-82 (1980)) or Brown et al. (Ann. Rev. Neurosci. 4:17-42 (1981));increased production of neuron-associated molecules may be measured bybioassay, enzymatic assay, antibody binding, Northern blot assay, etc.,using techniques known in the art and depending on the molecule to bemeasured; and motor neuron dysfunction may be measured by assessing thephysical manifestation of motor neuron disorder, e.g., weakness, motorneuron conduction velocity, or functional disability.

In specific embodiments, motor neuron diseases, disorders, and/orconditions that may be treated, prevented, and/or diagnosed according tothe invention include, but are not limited to, diseases, disorders,and/or conditions such as infarction, infection, exposure to toxin,trauma, surgical damage, degenerative disease or malignancy that mayaffect motor neurons as well as other components of the nervous system,as well as diseases, disorders, and/or conditions that selectivelyaffect neurons such as amyotrophic lateral sclerosis, and including, butnot limited to, progressive spinal muscular atrophy, progressive bulbarpalsy, primary lateral sclerosis, infantile and juvenile muscularatrophy, progressive bulbar paralysis of childhood (Fazio-Londesyndrome), poliomyelitis and the post polio syndrome, and HereditaryMotorsensory Neuropathy (Charcot-Marie-Tooth Disease).

Infectious Disease

A polypeptide or polynucleotide and/or agonist or antagonist of thepresent invention can be used to treat, prevent, and/or diagnoseinfectious agents. For example, by increasing the immune response,particularly increasing the proliferation and differentiation of Band/or T cells, infectious diseases may be treated, prevented, and/ordiagnosed. The immune response may be increased by either enhancing anexisting immune response, or by initiating a new immune response.Alternatively, polypeptide or polynucleotide and/or agonist orantagonist of the present invention may also directly inhibit theinfectious agent, without necessarily eliciting an immune response.

Viruses are one example of an infectious agent that can cause disease orsymptoms that can be treated, prevented, and/or diagnosed by apolynucleotide or polypeptide and/or agonist or antagonist of thepresent invention. Examples of viruses, include, but are not limited toExamples of viruses, include, but are not limited to the following DNAand RNA viruses and viral families: Arbovirus, Adenoviridae,Arenaviridae, Arterivirus, Birnaviridae, Bunyaviridae, Caliciviridae,Circoviridae, Coronaviridae, Dengue, EBV, HIV, Flaviviridae,Hepadnaviridae (Hepatitis), Herpesviridae (such as, Cytomegalovirus,Herpes Simplex, Herpes Zoster), Mononegavirus (e.g., Paramyxoviridae,Morbillivirus, Rhabdoviridae), Orthomyxoviridae (e.g., Influenza A,Influenza B, and parainfluenza), Papiloma virus, Papovaviridae,Parvoviridae, Picornaviridae, Poxyiridae (such as Smallpox or Vaccinia),Reoviridae (e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II,Lentivirus), and Togaviridae (e.g., Rubivirus). Viruses falling withinthese families can cause a variety of diseases or symptoms, including,but not limited to: arthritis, bronchiollitis, respiratory syncytialvirus, encephalitis, eye infections (e.g., conjunctivitis, keratitis),chronic fatigue syndrome, hepatitis (A, B, C, E, Chronic Active, Delta),Japanese B encephalitis, Junin, Chikungunya, Rift Valley fever, yellowfever, meningitis, opportunistic infections (e.g., AIDS), pneumonia,Burkitt's Lymphoma, chickenpox, hemorrhagic fever, Measles, Mumps,Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella,sexually transmitted diseases, skin diseases (e.g., Kaposi's, warts),and viremia. polynucleotides or polypeptides, or agonists or antagonistsof the invention, can be used to treat, prevent, and/or diagnose any ofthese symptoms or diseases. In specific embodiments, polynucleotides,polypeptides, or agonists or antagonists of the invention are used totreat, prevent, and/or diagnose: meningitis, Dengue, EBV, and/orhepatitis (e.g., hepatitis B). In an additional specific embodimentpolynucleotides, polypeptides, or agonists or antagonists of theinvention are used to treat patients nonresponsive to one or more othercommercially available hepatitis vaccines. In a further specificembodiment polynucleotides, polypeptides, or agonists or antagonists ofthe invention are used to treat, prevent, and/or diagnose AIDS.

Similarly, bacterial or fungal agents that can cause disease or symptomsand that can be treated, prevented, and/or diagnosed by a polynucleotideor polypeptide and/or agonist or antagonist of the present inventioninclude, but not limited to, include, but not limited to, the followingGram-Negative and Gram-positive bacteria and bacterial families andfungi: Actinomycetales (e.g., Corynebacterium, Mycobacterium,Norcardia), Cryptococcus neoformans, Aspergillosis, Bacillaceae (e.g.,Anthrax, Clostridium), Bacteroidaceae, Blastomycosis, Bordetella,Borrelia (e.g., Borrelia burgdorferi), Brucellosis, Candidiasis,Campylobacter, Coccidioidomycosis, Cryptococcosis, Dermatocycoses, E.coli (e.g., Enterotoxigenic E. coli and Enterohemorrhagic E. coli),Enterobacteriaceae (Klebsiella, Salmonella (e.g., Salmonella typhi, andSalmonella paratyphi), Serratia, Yersinia), Erysipelothrix,Helicobacter, Legionellosis, Leptospirosis, Listeria, Mycoplasmatales,Mycobacterium leprae, Vibrio cholerae, Neisseriaceae (e.g.,Acinetobacter, Gonorrhea, Menigococcal), Meisseria meningitidis,Pasteurellacea Infections (e.g., Actinobacillus, Heamophilus (e.g.,Heamophilus influenza type B), Pasteurella), Pseudomonas,Rickettsiaceae, Chlamydiaceae, Syphilis, Shigella spp., Staphylococcal,Meningiococcal, Pneumococcal and Streptococcal (e.g., Streptococcuspneumoniae and Group B Streptococcus). These bacterial or fungalfamilies can cause the following diseases or symptoms, including, butnot limited to: bacteremia, endocarditis, eye infections(conjunctivitis, tuberculosis, uveitis), gingivitis, opportunisticinfections (e.g., AIDS related infections), paronychia,prosthesis-related infections, Reiter's Disease, respiratory tractinfections, such as Whooping Cough or Empyema, sepsis, Lyme Disease,Cat-Scratch Disease, Dysentery, Paratyphoid Fever, food poisoning,Typhoid, pneumonia, Gonorrhea, meningitis (e.g., mengitis types A andB), Chlamydia, Syphilis, Diphtheria, Leprosy, Paratuberculosis,Tuberculosis, Lupus, Botulism, gangrene, tetanus, impetigo, RheumaticFever, Scarlet Fever, sexually transmitted diseases, skin diseases(e.g., cellulitis, dermatocycoses), toxemia, urinary tract infections,wound infections. Polynucleotides or polypeptides, agonists orantagonists of the invention, can be used to treat, prevent, and/ordiagnose any of these symptoms or diseases. In specific embodiments,polynucleotides, polypeptides, agonists or antagonists of the inventionare used to treat, prevent, and/or diagnose: tetanus, Diptheria,botulism, and/or meningitis type B.

Moreover, parasitic agents causing disease or symptoms that can betreated, prevented, and/or diagnosed by a polynucleotide or polypeptideand/or agonist or antagonist of the present invention include, but notlimited to, the following families or class: Amebiasis, Babesiosis,Coccidiosis, Cryptosporidiosis, Dientamoebiasis, Dourine, Ectoparasitic,Giardiasis, Helminthiasis, Leishmaniasis, Theileriasis, Toxoplasmosis,Trypanosomiasis, and Trichomonas and Sporozoans (e.g., Plasmodium virax,Plasmodium falciparium, Plasmodium malariae and Plasmodium ovale). Theseparasites can cause a variety of diseases or symptoms, including, butnot limited to: Scabies, Trombiculiasis, eye infections, intestinaldisease (e.g., dysentery, giardiasis), liver disease, lung disease,opportunistic infections (e.g., AIDS related), malaria, pregnancycomplications, and toxoplasmosis. polynucleotides or polypeptides, oragonists or antagonists of the invention, can be used to treat, prevent,and/or diagnose any of these symptoms or diseases. In specificembodiments, polynucleotides, polypeptides, or agonists or antagonistsof the invention are used to treat, prevent, and/or diagnose malaria.

Preferably, treatment or prevention using a polypeptide orpolynucleotide and/or agonist or antagonist of the present inventioncould either be by administering an effective amount of a polypeptide tothe patient, or by removing cells from the patient, supplying the cellswith a polynucleotide of the present invention, and returning theengineered cells to the patient (ex vivo therapy). Moreover, thepolypeptide or polynucleotide of the present invention can be used as anantigen in a vaccine to raise an immune response against infectiousdisease.

Regeneration

A polynucleotide or polypeptide and/or agonist or antagonist of thepresent invention can be used to differentiate, proliferate, and attractcells, leading to the regeneration of tissues. (See, Science 276:59-87(1997).) The regeneration of tissues could be used to repair, replace,or protect tissue damaged by congenital defects, trauma (wounds, burns,incisions, or ulcers), age, disease (e.g. osteoporosis, osteocarthritis,periodontal disease, liver failure), surgery, including cosmetic plasticsurgery, fibrosis, reperfusion injury, or systemic cytokine damage.

Tissues that could be regenerated using the present invention includeorgans (e.g., pancreas, liver, intestine, kidney, skin, endothelium),muscle (smooth, skeletal or cardiac), vasculature (including vascularand lymphatics), nervous, hematopoietic, and skeletal (bone, cartilage,tendon, and ligament) tissue. Preferably, regeneration occurs without ordecreased scarring. Regeneration also may include angiogenesis.

Moreover, a polynucleotide or polypeptide and/or agonist or antagonistof the present invention may increase regeneration of tissues difficultto heal. For example, increased tendon/ligament regeneration wouldquicken recovery time after damage. A polynucleotide or polypeptideand/or agonist or antagonist of the present invention could also be usedprophylactically in an effort to avoid damage. Specific diseases thatcould be treated, prevented, and/or diagnosed include of tendinitis,carpal tunnel syndrome, and other tendon or ligament defects. A furtherexample of tissue regeneration of non-healing wounds includes pressureulcers, ulcers associated with vascular insufficiency, surgical, andtraumatic wounds.

Similarly, nerve and brain tissue could also be regenerated by using apolynucleotide or polypeptide and/or agonist or antagonist of thepresent invention to proliferate and differentiate nerve cells. Diseasesthat could be treated, prevented, and/or diagnosed using this methodinclude central and peripheral nervous system diseases, neuropathies, ormechanical and traumatic diseases, disorders, and/or conditions (e.g.,spinal cord disorders, head trauma, cerebrovascular disease, and stoke).Specifically, diseases associated with peripheral nerve injuries,peripheral neuropathy (e.g., resulting from chemotherapy or othermedical therapies), localized neuropathies, and central nervous systemdiseases (e.g., Alzheimer's disease, Parkinson's disease, Huntington'sdisease, amyotrophic lateral sclerosis, and Shy-Drager syndrome), couldall be treated, prevented, and/or diagnosed using the polynucleotide orpolypeptide and/or agonist or antagonist of the present invention.

Chemotaxis

A polynucleotide or polypeptide and/or agonist or antagonist of thepresent invention may have chemotaxis activity. A chemotaxic moleculeattracts or mobilizes cells (e.g., monocytes, fibroblasts, neutrophils,T-cells, mast cells, eosinophils, epithelial and/or endothelial cells)to a particular site in the body, such as inflammation, infection, orsite of hyperproliferation. The mobilized cells can then fight offand/or heal the particular trauma or abnormality.

A polynucleotide or polypeptide and/or agonist or antagonist of thepresent invention may increase chemotaxic activity of particular cells.These chemotactic molecules can then be used to treat, prevent, and/ordiagnose inflammation, infection, hyperproliferative diseases,disorders, and/or conditions, or any immune system disorder byincreasing the number of cells targeted to a particular location in thebody. For example, chemotaxic molecules can be used to treat, prevent,and/or diagnose wounds and other trauma to tissues by attracting immunecells to the injured location. Chemotactic molecules of the presentinvention can also attract fibroblasts, which can be used to treat,prevent, and/or diagnose wounds.

It is also contemplated that a polynucleotide or polypeptide and/oragonist or antagonist of the present invention may inhibit chemotacticactivity. These molecules could also be used to treat, prevent, and/ordiagnose diseases, disorders, and/or conditions. Thus, a polynucleotideor polypeptide and/or agonist or antagonist of the present inventioncould be used as an inhibitor of chemotaxis.

Binding Activity

A polypeptide of the present invention may be used to screen formolecules that bind to the polypeptide or for molecules to which thepolypeptide binds. The binding of the polypeptide and the molecule mayactivate (agonist), increase, inhibit (antagonist), or decrease activityof the polypeptide or the molecule bound. Examples of such moleculesinclude antibodies, oligonucleotides, proteins (e.g., receptors), orsmall molecules.

Preferably, the molecule is closely related to the natural ligand of thepolypeptide, e.g., a fragment of the ligand, or a natural substrate, aligand, a structural or functional mimetic. (See, Coligan et al.,Current Protocols in Immunology 1(2):Chapter 5 (1991).) Similarly, themolecule can be closely related to the natural receptor to which thepolypeptide binds, or at least, a fragment of the receptor capable ofbeing bound by the polypeptide (e.g., active site). In either case, themolecule can be rationally designed using known techniques.

Preferably, the screening for these molecules involves producingappropriate cells which express the polypeptide, either as a secretedprotein or on the cell membrane. Preferred cells include cells frommammals, yeast, Drosophila, or E. coli. Cells expressing the polypeptide(or cell membrane containing the expressed polypeptide) are thenpreferably contacted with a test compound potentially containing themolecule to observe binding, stimulation, or inhibition of activity ofeither the polypeptide or the molecule.

The assay may simply test binding of a candidate compound to thepolypeptide, wherein binding is detected by a label, or in an assayinvolving competition with a labeled competitor. Further, the assay maytest whether the candidate compound results in a signal generated bybinding to the polypeptide.

Alternatively, the assay can be carried out using cell-freepreparations, polypeptide/molecule affixed to a solid support, chemicallibraries, or natural product mixtures. The assay may also simplycomprise the steps of mixing a candidate compound with a solutioncontaining a polypeptide, measuring polypeptide/molecule activity orbinding, and comparing the polypeptide/molecule activity or binding to astandard.

Preferably, an ELISA assay can measure polypeptide level or activity ina sample (e.g., biological sample) using a monoclonal or polyclonalantibody. The antibody can measure polypeptide level or activity byeither binding, directly or indirectly, to the polypeptide or bycompeting with the polypeptide for a substrate.

Additionally, the receptor to which a polypeptide of the invention bindscan be identified by numerous methods known to those of skill in theart, for example, ligand panning and FACS sorting (Coligan, et al.,Current Protocols in Immun., 1(2), Chapter 5, (1991)). For example,expression cloning is employed wherein polyadenylated RNA is preparedfrom a cell responsive to the polypeptides, for example, NIH3T3 cellswhich are known to contain multiple receptors for the FGF familyproteins, and SC-3 cells, and a cDNA library created from this RNA isdivided into pools and used to transfect COS cells or other cells thatare not responsive to the polypeptides. Transfected cells which aregrown on glass slides are exposed to the polypeptide of the presentinvention, after they have been labeled. The polypeptides can be labeledby a variety of means including iodination or inclusion of a recognitionsite for a site-specific protein kinase.

Following fixation and incubation, the slides are subjected toauto-radiographic analysis. Positive pools are identified and sub-poolsare prepared and re-transfected using an iterative sub-pooling andre-screening process, eventually yielding a single clones that encodesthe putative receptor.

As an alternative approach for receptor identification, the labeledpolypeptides can be photoaffinity linked with cell membrane or extractpreparations that express the receptor molecule. Cross-linked materialis resolved by PAGE analysis and exposed to X-ray film. The labeledcomplex containing the receptors of the polypeptides can be excised,resolved into peptide fragments, and subjected to proteinmicrosequencing. The amino acid sequence obtained from microsequencingwould be used to design a set of degenerate oligonucleotide probes toscreen a cDNA library to identify the genes encoding the putativereceptors.

Moreover, the techniques of gene-shuffling, motif-shuffling,exon-shuffling, and/or codon-shuffling (collectively referred to as “DNAshuffling”) may be employed to modulate the activities of polypeptidesof the invention thereby effectively generating agonists and antagonistsof polypeptides of the invention. See generally, U.S. Pat. Nos.5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458, and Patten,P. A., et al., Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama, S.Trends Biotechnol. 16(2):76-82 (1998); Hansson, L. O., et al., J. Mol.Biol. 287:265-76 (1999); and Lorenzo, M. M. and Blasco, R. Biotechniques24(2):308-13 (1998) (each of these patents and publications are herebyincorporated by reference). In one embodiment, alteration ofpolynucleotides and corresponding polypeptides of the invention may beachieved by DNA shuffling. DNA shuffling involves the assembly of two ormore DNA segments into a desired polynucleotide sequence of theinvention molecule by homologous, or site-specific, recombination. Inanother embodiment, polynucleotides and corresponding polypeptides ofthe invention may be altered by being subjected to random mutagenesis byerror-prone PCR, random nucleotide insertion or other methods prior torecombination. In another embodiment, one or more components, motifs,sections, parts, domains, fragments, etc., of the polypeptides of theinvention may be recombined with one or more components, motifs,sections, parts, domains, fragments, etc. of one or more heterologousmolecules. In preferred embodiments, the heterologous molecules arefamily members. In further preferred embodiments, the heterologousmolecule is a growth factor such as, for example, platelet-derivedgrowth factor (PDGF), insulin-like growth factor (IGF-1), transforminggrowth factor (TGF)-alpha, epidermal growth factor (EGF), fibroblastgrowth factor (FGF), TGF-beta, bone morphogenetic protein (BMP)-2,BMP-4, BMP-5, BMP-6, BMP-7, activins A and B, decapentaplegic (dpp),60A, OP-2, dorsalin, growth differentiation factors (GDFs), nodal, MIS,inhibin-alpha, TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta5, andglial-derived neurotrophic factor (GDNF).

Other preferred fragments are biologically active fragments of thepolypeptides of the invention. Biologically active fragments are thoseexhibiting activity similar, but not necessarily identical, to anactivity of the polypeptide. The biological activity of the fragmentsmay include an improved desired activity, or a decreased undesirableactivity.

Additionally, this invention provides a method of screening compounds toidentify those which modulate the action of the polypeptide of thepresent invention. An example of such an assay comprises combining amammalian fibroblast cell, a the polypeptide of the present invention,the compound to be screened and 3[H] thymidine under cell cultureconditions where the fibroblast cell would normally proliferate. Acontrol assay may be performed in the absence of the compound to bescreened and compared to the amount of fibroblast proliferation in thepresence of the compound to determine if the compound stimulatesproliferation by determining the uptake of 3[H] thymidine in each case.The amount of fibroblast cell proliferation is measured by liquidscintillation chromatography which measures the incorporation of 3[H]thymidine. Both agonist and antagonist compounds may be identified bythis procedure.

In another method, a mammalian cell or membrane preparation expressing areceptor for a polypeptide of the present invention is incubated with alabeled polypeptide of the present invention in the presence of thecompound. The ability of the compound to enhance or block thisinteraction could then be measured. Alternatively, the response of aknown second messenger system following interaction of a compound to bescreened and the receptor is measured and the ability of the compound tobind to the receptor and elicit a second messenger response is measuredto determine if the compound is a potential agonist or antagonist. Suchsecond messenger systems include but are not limited to, cAMP guanylatecyclase, ion channels or phosphoinositide hydrolysis.

All of these above assays can be used as diagnostic or prognosticmarkers. The molecules discovered using these assays can be used totreat, prevent, and/or diagnose disease or to bring about a particularresult in a patient (e.g., blood vessel growth) by activating orinhibiting the polypeptide/molecule. Moreover, the assays can discoveragents which may inhibit or enhance the production of the polypeptidesof the invention from suitably manipulated cells or tissues. Therefore,the invention includes a method of identifying compounds which bind tothe polypeptides of the invention comprising the steps of: (a)incubating a candidate binding compound with the polypeptide; and (b)determining if binding has occurred. Moreover, the invention includes amethod of identifying agonists/antagonists comprising the steps of: (a)incubating a candidate compound with the polypeptide, (b) assaying abiological activity, and (b) determining if a biological activity of thepolypeptide has been altered.

Also, one could identify molecules bind a polypeptide of the inventionexperimentally by using the beta-pleated sheet regions contained in thepolypeptide sequence of the protein. Accordingly, specific embodimentsof the invention are directed to polynucleotides encoding polypeptideswhich comprise, or alternatively consist of, the amino acid sequence ofeach beta pleated sheet regions in a disclosed polypeptide sequence.Additional embodiments of the invention are directed to polynucleotidesencoding polypeptides which comprise, or alternatively consist of, anycombination or all of contained in the polypeptide sequences of theinvention. Additional preferred embodiments of the invention aredirected to polypeptides which comprise, or alternatively consist of,the amino acid sequence of each of the beta pleated sheet regions in oneof the polypeptide sequences of the invention. Additional embodiments ofthe invention are directed to polypeptides which comprise, oralternatively consist of, any combination or all of the beta pleatedsheet regions in one of the polypeptide sequences of the invention.

Targeted Delivery

In another embodiment, the invention provides a method of deliveringcompositions to targeted cells expressing a receptor for a polypeptideof the invention, or cells expressing a cell bound form of a polypeptideof the invention.

As discussed herein, polypeptides or antibodies of the invention may beassociated with heterologous polypeptides, heterologous nucleic acids,toxins, or prodrugs via hydrophobic, hydrophilic, ionic and/or covalentinteractions. In one embodiment, the invention provides a method for thespecific delivery of compositions of the invention to cells byadministering polypeptides of the invention (including antibodies) thatare associated with heterologous polypeptides or nucleic acids. In oneexample, the invention provides a method for delivering a therapeuticprotein into the targeted cell. In another example, the inventionprovides a method for delivering a single stranded nucleic acid (e.g.,antisense or ribozymes) or double stranded nucleic acid (e.g., DNA thatcan integrate into the cell's genome or replicate episomally and thatcan be transcribed) into the targeted cell.

In another embodiment, the invention provides a method for the specificdestruction of cells (e.g., the destruction of tumor cells) byadministering polypeptides of the invention (e.g., polypeptides of theinvention or antibodies of the invention) in association with toxins orcytotoxic prodrugs.

By “toxin” is meant compounds that bind and activate endogenouscytotoxic effector systems, radioisotopes, holotoxins, modified toxins,catalytic subunits of toxins, or any molecules or enzymes not normallypresent in or on the surface of a cell that under defined conditionscause the cell's death. Toxins that may be used according to the methodsof the invention include, but are not limited to, radioisotopes known inthe art, compounds such as, for example, antibodies (or complementfixing containing portions thereof) that bind an inherent or inducedendogenous cytotoxic effector system, thymidine kinase, endonuclease,RNAse, alpha toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheriatoxin, saporin, momordin, gelonin, pokeweed antiviral protein,alpha-sarcin and cholera toxin. By “cytotoxic prodrug” is meant anon-toxic compound that is converted by an enzyme, normally present inthe cell, into a cytotoxic compound. Cytotoxic prodrugs that may be usedaccording to the methods of the invention include, but are not limitedto, glutamyl derivatives of benzoic acid mustard alkylating agent,phosphate derivatives of etoposide or mitomycin C, cytosine arabinoside,daunorubisin, and phenoxyacetamide derivatives of doxorubicin.

Drug Screening

Further contemplated is the use of the polypeptides of the presentinvention, or the polynucleotides encoding these polypeptides, to screenfor molecules which modify the activities of the polypeptides of thepresent invention. Such a method would include contacting thepolypeptide of the present invention with a selected compound(s)suspected of having antagonist or agonist activity, and assaying theactivity of these polypeptides following binding.

This invention is particularly useful for screening therapeuticcompounds by using the polypeptides of the present invention, or bindingfragments thereof, in any of a variety of drug screening techniques. Thepolypeptide or fragment employed in such a test may be affixed to asolid support, expressed on a cell surface, free in solution, or locatedintracellularly. One method of drug screening utilizes eukaryotic orprokaryotic host cells which are stably transformed with recombinantnucleic acids expressing the polypeptide or fragment. Drugs are screenedagainst such transformed cells in competitive binding assays. One maymeasure, for example, the formulation of complexes between the agentbeing tested and a polypeptide of the present invention.

Thus, the present invention provides methods of screening for drugs orany other agents which affect activities mediated by the polypeptides ofthe present invention. These methods comprise contacting such an agentwith a polypeptide of the present invention or a fragment thereof andassaying for the presence of a complex between the agent and thepolypeptide or a fragment thereof, by methods well known in the art. Insuch a competitive binding assay, the agents to screen are typicallylabeled. Following incubation, free agent is separated from that presentin bound form, and the amount of free or uncomplexed label is a measureof the ability of a particular agent to bind to the polypeptides of thepresent invention.

Another technique for drug screening provides high throughput screeningfor compounds having suitable binding affinity to the polypeptides ofthe present invention, and is described in great detail in EuropeanPatent Application 84/03564, published on Sep. 13, 1984, which isincorporated herein by reference herein. Briefly stated, large numbersof different small peptide test compounds are synthesized on a solidsubstrate, such as plastic pins or some other surface. The peptide testcompounds are reacted with polypeptides of the present invention andwashed. Bound polypeptides are then detected by methods well known inthe art. Purified polypeptides are coated directly onto plates for usein the aforementioned drug screening techniques. In addition,non-neutralizing antibodies may be used to capture the peptide andimmobilize it on the solid support.

This invention also contemplates the use of competitive drug screeningassays in which neutralizing antibodies capable of binding polypeptidesof the present invention specifically compete with a test compound forbinding to the polypeptides or fragments thereof. In this manner, theantibodies are used to detect the presence of any peptide which sharesone or more antigenic epitopes with a polypeptide of the invention.

The human phosphatase polypeptides and/or peptides of the presentinvention, or immunogenic fragments or oligopeptides thereof, can beused for screening therapeutic drugs or compounds in a variety of drugscreening techniques. The fragment employed in such a screening assaymay be free in solution, affixed to a solid support, borne on a cellsurface, or located intracellularly. The reduction or abolition ofactivity of the formation of binding complexes between the ion channelprotein and the agent being tested can be measured. Thus, the presentinvention provides a method for screening or assessing a plurality ofcompounds for their specific binding affinity with a phosphatasepolypeptide, or a bindable peptide fragment, of this invention,comprising providing a plurality of compounds, combining the phosphatasepolypeptide, or a bindable peptide fragment, with each of a plurality ofcompounds for a time sufficient to allow binding under suitableconditions and detecting binding of the phosphatase polypeptide orpeptide to each of the plurality of test compounds, thereby identifyingthe compounds that specifically bind to the phosphatase polypeptide orpeptide.

Methods of identifying compounds that modulate the activity of the novelhuman phosphatase polypeptides and/or peptides are provided by thepresent invention and comprise combining a potential or candidatecompound or drug modulator of phosphatase activity with a phosphatasepolypeptide or peptide, for example, the phosphatase amino acid sequenceas set forth in SEQ ID NO:42, 109, 150, or 152, and measuring an effectof the candidate compound or drug modulator on the biological activityof the phosphatase polypeptide or peptide. Such measurable effectsinclude, for example, physical binding interaction; the ability tophosphorylate a suitable calpain substrate; effects on native and clonedphosphatase-expressing cell line; and effects of modulators or otherphosphatase-mediated physiological measures.

Another method of identifying compounds that modulate the biologicalactivity of the novel phosphatase polypeptides of the present inventioncomprises combining a potential or candidate compound or drug modulatorof a phosphatase activity with a host cell that expresses thephosphatase polypeptide and measuring an effect of the candidatecompound or drug modulator on the biological activity of the phosphatasepolypeptide. The host cell can also be capable of being induced toexpress the phosphatase polypeptide, e.g., via inducible expression.Physiological effects of a given modulator candidate on the phosphatasepolypeptide can also be measured. Thus, cellular assays for particularphosphatase modulators may be either direct measurement orquantification of the physical biological activity of the phosphatasepolypeptide, or they may be measurement or quantification of aphysiological effect. Such methods preferably employ a phosphatasepolypeptide as described herein, or an overexpressed recombinantphosphatase polypeptide in suitable host cells containing an expressionvector as described herein, wherein the phosphatase polypeptide isexpressed, overexpressed, or undergoes upregulated expression.

Another aspect of the present invention embraces a method of screeningfor a compound that is capable of modulating the biological activity ofa phosphatase polypeptide, comprising providing a host cell containingan expression vector harboring a nucleic acid sequence encoding aphosphatase polypeptide, or a functional peptide or portion thereof(e.g., SEQ ID NO:42, 109, 150, or 152); determining the biologicalactivity of the expressed phosphatase polypeptide in the absence of amodulator compound; contacting the cell with the modulator compound anddetermining the biological activity of the expressed phosphatasepolypeptide in the presence of the modulator compound. In such a method,a difference between the activity of the phosphatase polypeptide in thepresence of the modulator compound and in the absence of the modulatorcompound indicates a modulating effect of the compound.

Essentially any chemical compound can be employed as a potentialmodulator or ligand in the assays according to the present invention.Compounds tested as phosphatase modulators can be any small chemicalcompound, or biological entity (e.g., protein, sugar, nucleic acid,lipid). Test compounds will typically be small chemical molecules andpeptides. Generally, the compounds used as potential modulators can bedissolved in aqueous or organic (e.g., DMSO-based) solutions. The assaysare designed to screen large chemical libraries by automating the assaysteps and providing compounds from any convenient source. Assays aretypically run in parallel, for example, in microtiter formats onmicrotiter plates in robotic assays. There are many suppliers ofchemical compounds, including Sigma (St. Louis, Mo.), Aldrich (St.Louis, Mo.), Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-BiochemicaAnalytika (Buchs, Switzerland), for example. Also, compounds may besynthesized by methods known in the art.

High throughput screening methodologies are particularly envisioned forthe detection of modulators of the novel phosphatase polynucleotides andpolypeptides described herein. Such high throughput screening methodstypically involve providing a combinatorial chemical or peptide librarycontaining a large number of potential therapeutic compounds (e.g.,ligand or modulator compounds). Such combinatorial chemical libraries orligand libraries are then screened in one or more assays to identifythose library members (e.g., particular chemical species or subclasses)that display a desired characteristic activity. The compounds soidentified can serve as conventional lead compounds, or can themselvesbe used as potential or actual therapeutics.

A combinatorial chemical library is a collection of diverse chemicalcompounds generated either by chemical synthesis or biologicalsynthesis, by combining a number of chemical building blocks (i.e.,reagents such as amino acids). As an example, a linear combinatoriallibrary, e.g., a polypeptide or peptide library, is formed by combininga set of chemical building blocks in every possible way for a givencompound length (i.e., the number of amino acids in a polypeptide orpeptide compound). Millions of chemical compounds can be synthesizedthrough such combinatorial mixing of chemical building blocks.

The preparation and screening of combinatorial chemical libraries iswell known to those having skill in the pertinent art. Combinatoriallibraries include, without limitation, peptide libraries (e.g. U.S. Pat.No. 5,010,175; Furka, 1991, Int. J. Pept. Prot. Res., 37:487-493; andHoughton et al., 1991, Nature, 354:84-88). Other chemistries forgenerating chemical diversity libraries can also be used. Nonlimitingexamples of chemical diversity library chemistries include, peptides(PCT Publication No. WO 91/019735), encoded peptides (PCT PublicationNo. WO 93/20242), random bio-oligomers (PCT Publication No. WO92/00091), benzodiazepines (U.S. Pat. No. 5,288,514), diversomers suchas hydantoins, benzodiazepines and dipeptides (Hobbs et al., 1993, Proc.Natl. Acad. Sci. USA, 90:6909-6913), vinylogous polypeptides (Hagiharaet al., 1992, J. Amer. Chem. Soc., 114:6568), nonpeptidalpeptidomimetics with glucose scaffolding (Hirschmann et al., 1992, J.Amer. Chem. Soc., 114:9217-9218), analogous organic synthesis of smallcompound libraries (Chen et al., 1994, J. Amer. Chem. Soc., 116:2661),oligocarbamates (Cho et al., 1993, Science, 261:1303), and/or peptidylphosphonates (Campbell et al., 1994, J. Org. Chem., 59:658), nucleicacid libraries (see Ausubel, Berger and Sambrook, all supra), peptidenucleic acid libraries (U.S. Pat. No. 5,539,083), antibody libraries(e.g., Vaughn et al., 1996, Nature Biotechnology, 14(3):309-314) andPCT/US96/10287), carbohydrate libraries (e.g., Liang et al., 1996,Science, 274-1520-1522) and U.S. Pat. No. 5,593,853), small organicmolecule libraries (e.g., benzodiazepines, Baum C&EN, Jan. 18, 1993,page 33; and U.S. Pat. No. 5,288,514; isoprenoids, U.S. Pat. No.5,569,588; thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974;pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholinocompounds, U.S. Pat. No. 5,506,337; and the like).

Devices for the preparation of combinatorial libraries are commerciallyavailable (e.g., 357 MPS, 390 MPS, Advanced Chem Tech, Louisville Ky.;Symphony, Rainin, Woburn, Mass.; 433A Applied Biosystems, Foster City,Calif.; 9050 Plus, Millipore, Bedford, Mass.). In addition, a largenumber of combinatorial libraries are commercially available (e.g.,ComGenex, Princeton, N.J.; Asinex, Moscow, Russia; Tripos, Inc., St.Louis, Mo.; ChemStar, Ltd., Moscow, Russia; 3D Pharmaceuticals, Exton,Pa.; Martek Biosciences, Columbia, Md., and the like).

In one embodiment, the invention provides solid phase based in vitroassays in a high throughput format, where the cell or tissue expressingan ion channel is attached to a solid phase substrate. In such highthroughput assays, it is possible to screen up to several thousanddifferent modulators or ligands in a single day. In particular, eachwell of a microtiter plate can be used to perform a separate assayagainst a selected potential modulator, or, if concentration orincubation time effects are to be observed, every 5-10 wells can test asingle modulator. Thus, a single standard microtiter plate can assayabout 96 modulators. If 1536 well plates are used, then a single platecan easily assay from about 100 to about 1500 different compounds. It ispossible to assay several different plates per day; thus, for example,assay screens for up to about 6,000-20,000 different compounds arepossible using the described integrated systems.

In another of its aspects, the present invention encompasses screeningand small molecule (e.g., drug) detection assays which involve thedetection or identification of small molecules that can bind to a givenprotein, i.e., a phosphatase polypeptide or peptide. Particularlypreferred are assays suitable for high throughput screeningmethodologies.

In such binding-based detection, identification, or screening assays, afunctional assay is not typically required. All that is needed is atarget protein, preferably substantially purified, and a library orpanel of compounds (e.g., ligands, drugs, small molecules) or biologicalentities to be screened or assayed for binding to the protein target.Preferably, most small molecules that bind to the target protein willmodulate activity in some manner, due to preferential, higher affinitybinding to functional areas or sites on the protein.

An example of such an assay is the fluorescence based thermal shiftassay (3-Dimensional Pharmaceuticals, Inc., 3DP, Exton, Pa.) asdescribed in U.S. Pat. Nos. 6,020,141 and 6,036,920 to Pantoliano etal.; see also, J. Zimmerman, 2000, Gen. Eng. News, 20(8)). The assayallows the detection of small molecules (e.g., drugs, ligands) that bindto expressed, and preferably purified, ion channel polypeptide based onaffinity of binding determinations by analyzing thermal unfolding curvesof protein-drug or ligand complexes. The drugs or binding moleculesdetermined by this technique can be further assayed, if desired, bymethods, such as those described herein, to determine if the moleculesaffect or modulate function or activity of the target protein.

To purify a phosphatase polypeptide or peptide to measure a biologicalbinding or ligand binding activity, the source may be a whole celllysate that can be prepared by successive freeze-thaw cycles (e.g., oneto three) in the presence of standard protease inhibitors. Thephosphatase polypeptide may be partially or completely purified bystandard protein purification methods, e.g., affinity chromatographyusing specific antibody described infra, or by ligands specific for anepitope tag engineered into the recombinant phosphatase polypeptidemolecule, also as described herein. Binding activity can then bemeasured as described.

Compounds which are identified according to the methods provided herein,and which modulate or regulate the biological activity or physiology ofthe phosphatase polypeptides according to the present invention are apreferred embodiment of this invention. It is contemplated that suchmodulatory compounds may be employed in treatment and therapeuticmethods for treating a condition that is mediated by the novelphosphatase polypeptides by administering to an individual in need ofsuch treatment a therapeutically effective amount of the compoundidentified by the methods described herein.

In addition, the present invention provides methods for treating anindividual in need of such treatment for a disease, disorder, orcondition that is mediated by the phosphatase polypeptides of theinvention, comprising administering to the individual a therapeuticallyeffective amount of the phosphatase-modulating compound identified by amethod provided herein.

Antisense and Ribozyme (Antagonists)

In specific embodiments, antagonists according to the present inventionare nucleic acids corresponding to the sequences contained in SEQ IDNO:X, or the complementary strand thereof, and/or to nucleotidesequences contained a deposited clone. In one embodiment, antisensesequence is generated internally by the organism, in another embodiment,the antisense sequence is separately administered (see, for example,O'Connor, Neurochem., 56:560 (1991). Oligodeoxynucleotides as AntisenseInhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988).Antisense technology can be used to control gene expression throughantisense DNA or RNA, or through triple-helix formation. Antisensetechniques are discussed for example, in Okano, Neurochem., 56:560(1991); Oligodeoxynucleotides as Antisense Inhibitors of GeneExpression, CRC Press, Boca Raton, Fla. (1988). Triple helix formationis discussed in, for instance, Lee et al., Nucleic Acids Research,6:3073 (1979); Cooney et al., Science, 241:456 (1988); and Dervan etal., Science, 251:1300 (1991). The methods are based on binding of apolynucleotide to a complementary DNA or RNA.

For example, the use of c-myc and c-myb antisense RNA constructs toinhibit the growth of the non-lymphocytic leukemia cell line HL-60 andother cell lines was previously described. (Wickstrom et al. (1988);Anfossi et al. (1989)). These experiments were performed in vitro byincubating cells with the oligoribonucleotide. A similar procedure forin vivo use is described in WO 91/15580. Briefly, a pair ofoligonucleotides for a given antisense RNA is produced as follows: Asequence complimentary to the first 15 bases of the open reading frameis flanked by an EcoR1 site on the 5 end and a HindIII site on the 3end. Next, the pair of oligonucleotides is heated at 90° C. for oneminute and then annealed in 2× ligation buffer (20 mM TRIS HCl pH 7.5,10 mM MgCl2, 10 mM dithiothreitol (DTT) and 0.2 mM ATP) and then ligatedto the EcoRI/Hind III site of the retroviral vector PMV7 (WO 91/15580).

For example, the 5′ coding portion of a polynucleotide that encodes themature polypeptide of the present invention may be used to design anantisense RNA oligonucleotide of from about 10 to 40 base pairs inlength. A DNA oligonucleotide is designed to be complementary to aregion of the gene involved in transcription thereby preventingtranscription and the production of the receptor. The antisense RNAoligonucleotide hybridizes to the mRNA in vivo and blocks translation ofthe mRNA molecule into receptor polypeptide.

In one embodiment, the antisense nucleic acid of the invention isproduced intracellularly by transcription from an exogenous sequence.For example, a vector or a portion thereof, is transcribed, producing anantisense nucleic acid (RNA) of the invention. Such a vector wouldcontain a sequence encoding the antisense nucleic acid of the invention.Such a vector can remain episomal or become chromosomally integrated, aslong as it can be transcribed to produce the desired antisense RNA. Suchvectors can be constructed by recombinant DNA technology methodsstandard in the art. Vectors can be plasmid, viral, or others known inthe art, used for replication and expression in vertebrate cells.Expression of the sequence encoding a polypeptide of the invention, orfragments thereof, can be by any promoter known in the art to act invertebrate, preferably human cells. Such promoters can be inducible orconstitutive. Such promoters include, but are not limited to, the SV40early promoter region (Bernoist and Chambon, Nature, 29:304-310 (1981),the promoter contained in the 3′ long terminal repeat of Rous sarcomavirus (Yamamoto et al., Cell, 22:787-797 (1980), the herpes thymidinepromoter (Wagner et al., Proc. Natl. Acad. Sci. U.S.A., 78:1441-1445(1981), the regulatory sequences of the metallothionein gene (Brinsteret al., Nature, 296:39-42 (1982)), etc.

The antisense nucleic acids of the invention comprise a sequencecomplementary to at least a portion of an RNA transcript of a gene ofinterest. However, absolute complementarity, although preferred, is notrequired. A sequence “complementary to at least a portion of an RNA,”referred to herein, means a sequence having sufficient complementarityto be able to hybridize with the RNA, forming a stable duplex; in thecase of double stranded antisense nucleic acids of the invention, asingle strand of the duplex DNA may thus be tested, or triplex formationmay be assayed. The ability to hybridize will depend on both the degreeof complementarity and the length of the antisense nucleic acidGenerally, the larger the hybridizing nucleic acid, the more basemismatches with a RNA sequence of the invention it may contain and stillform a stable duplex (or triplex as the case may be). One skilled in theart can ascertain a tolerable degree of mismatch by use of standardprocedures to determine the melting point of the hybridized complex.

Oligonucleotides that are complementary to the 5′ end of the message,e.g., the 5′ untranslated sequence up to and including the AUGinitiation codon, should work most efficiently at inhibitingtranslation. However, sequences complementary to the 3′ untranslatedsequences of mRNAs have been shown to be effective at inhibitingtranslation of mRNAs as well. See generally, Wagner, R., Nature,372:333-335 (1994). Thus, oligonucleotides complementary to either the5′- or 3′-non-translated, non-coding regions of a polynucleotidesequence of the invention could be used in an antisense approach toinhibit translation of endogenous mRNA. Oligonucleotides complementaryto the 5′ untranslated region of the mRNA should include the complementof the AUG start codon. Antisense oligonucleotides complementary to mRNAcoding regions are less efficient inhibitors of translation but could beused in accordance with the invention. Whether designed to hybridize tothe 5′-, 3′- or coding region of mRNA, antisense nucleic acids should beat least six nucleotides in length, and are preferably oligonucleotidesranging from 6 to about 50 nucleotides in length. In specific aspectsthe oligonucleotide is at least 10 nucleotides, at least 17 nucleotides,at least 25 nucleotides or at least 50 nucleotides.

The polynucleotides of the invention can be DNA or RNA or chimericmixtures or derivatives or modified versions thereof, single-stranded ordouble-stranded. The oligonucleotide can be modified at the base moiety,sugar moiety, or phosphate backbone, for example, to improve stabilityof the molecule, hybridization, etc. The oligonucleotide may includeother appended groups such as peptides (e.g., for targeting host cellreceptors in vivo), or agents facilitating transport across the cellmembrane (see, e.g., Letsinger et al., Proc. Natl. Acad. Sci. U.S.A.86:6553-6556 (1989); Lemaitre et al., Proc. Natl. Acad. Sci., 84:648-652(1987); PCT Publication NO: WO88/09810, published Dec. 15, 1988) or theblood-brain barrier (see, e.g., PCT Publication NO: WO89/10134,published Apr. 25, 1988), hybridization-triggered cleavage agents. (See,e.g., Krol et al., BioTechniques, 6:958-976 (1988)) or intercalatingagents. (See, e.g., Zon, Pharm. Res., 5:539-549 (1988)). To this end,the oligonucleotide may be conjugated to another molecule, e.g., apeptide, hybridization triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

The antisense oligonucleotide may comprise at least one modified basemoiety which is selected from the group including, but not limited to,5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

The antisense oligonucleotide may also comprise at least one modifiedsugar moiety selected from the group including, but not limited to,arabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the antisense oligonucleotide comprises atleast one modified phosphate backbone selected from the group including,but not limited to, a phosphorothioate, a phosphorodithioate, aphosphoramidothioate, a phosphoramidate, a phosphordiamidate, amethylphosphonate, an alkyl phosphotriester, and a formacetal or analogthereof.

In yet another embodiment, the antisense oligonucleotide is ana-anomeric oligonucleotide. An a-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual b-units, the strands run parallel to each other (Gautier et al.,Nucl. Acids Res., 15:6625-6641 (1987)). The oligonucleotide is a2-O-methylribonucleotide (Inoue et al., Nucl. Acids Res., 15:6131-6148(1987)), or a chimeric RNA-DNA analogue (Inoue et al., FEBS Lett.215:327-330 (1987)).

Polynucleotides of the invention may be synthesized by standard methodsknown in the art, e.g. by use of an automated DNA synthesizer (such asare commercially available from Biosearch, Applied Biosystems, etc.). Asexamples, phosphorothioate oligonucleotides may be synthesized by themethod of Stein et al. (Nucl. Acids Res., 16:3209 (1988)),methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., Proc. Natl. Acad. Sci.U.S.A., 85:7448-7451 (1988)), etc.

While antisense nucleotides complementary to the coding region sequenceof the invention could be used, those complementary to the transcribeduntranslated region are most preferred.

Potential antagonists according to the invention also include catalyticRNA, or a ribozyme (See, e.g., PCT International Publication WO90/11364, published Oct. 4, 1990; Sarver et al, Science, 247:1222-1225(1990). While ribozymes that cleave mRNA at site specific recognitionsequences can be used to destroy mRNAs corresponding to thepolynucleotides of the invention, the use of hammerhead ribozymes ispreferred. Hammerhead ribozymes cleave mRNAs at locations dictated byflanking regions that form complementary base pairs with the targetmRNA. The sole requirement is that the target mRNA have the followingsequence of two bases: 5′-UG-3′. The construction and production ofhammerhead ribozymes is well known in the art and is described morefully in Haseloff and Gerlach, Nature, 334:585-591 (1988). There arenumerous potential hammerhead ribozyme cleavage sites within eachnucleotide sequence disclosed in the sequence listing. Preferably, theribozyme is engineered so that the cleavage recognition site is locatednear the 5′ end of the mRNA corresponding to the polynucleotides of theinvention; i.e., to increase efficiency and minimize the intracellularaccumulation of non-functional mRNA transcripts.

As in the antisense approach, the ribozymes of the invention can becomposed of modified oligonucleotides (e.g. for improved stability,targeting, etc.) and should be delivered to cells which express thepolynucleotides of the invention in vivo. DNA constructs encoding theribozyme may be introduced into the cell in the same manner as describedabove for the introduction of antisense encoding DNA. A preferred methodof delivery involves using a DNA construct “encoding” the ribozyme underthe control of a strong constitutive promoter, such as, for example, polIII or pol II promoter, so that transfected cells will producesufficient quantities of the ribozyme to destroy endogenous messages andinhibit translation. Since ribozymes unlike antisense molecules, arecatalytic, a lower intracellular concentration is required forefficiency.

Antagonist/agonist compounds may be employed to inhibit the cell growthand proliferation effects of the polypeptides of the present inventionon neoplastic cells and tissues, i.e. stimulation of angiogenesis oftumors, and, therefore, retard or prevent abnormal cellular growth andproliferation, for example, in tumor formation or growth.

The antagonist/agonist may also be employed to prevent hyper-vasculardiseases, and prevent the proliferation of epithelial lens cells afterextracapsular cataract surgery. Prevention of the mitogenic activity ofthe polypeptides of the present invention may also be desirous in casessuch as restenosis after balloon angioplasty.

The antagonist/agonist may also be employed to prevent the growth ofscar tissue during wound healing.

The antagonist/agonist may also be employed to treat, prevent, and/ordiagnose the diseases described herein.

Thus, the invention provides a method of treating or preventingdiseases, disorders, and/or conditions, including but not limited to thediseases, disorders, and/or conditions listed throughout thisapplication, associated with overexpression of a polynucleotide of thepresent invention by administering to a patient (a) an antisensemolecule directed to the polynucleotide of the present invention, and/or(b) a ribozyme directed to the polynucleotide of the present invention.

invention, and/or (b) a ribozyme directed to the polynucleotide of thepresent invention.

Biotic Associations

A polynucleotide or polypeptide and/or agonist or antagonist of thepresent invention may increase the organisms ability, either directly orindirectly, to initiate and/or maintain biotic associations with otherorganisms. Such associations may be symbiotic, nonsymbiotic,endosymbiotic, macrosymbiotic, and/or microsymbiotic in nature. Ingeneral, a polynucleotide or polypeptide and/or agonist or antagonist ofthe present invention may increase the organisms ability to form bioticassociations with any member of the fungal, bacterial, lichen,mycorrhizal, cyanobacterial, dinoflaggellate, and/or algal, kingdom,phylums, families, classes, genuses, and/or species.

The mechanism by which a polynucleotide or polypeptide and/or agonist orantagonist of the present invention may increase the host organismsability, either directly or indirectly, to initiate and/or maintainbiotic associations is variable, though may include, modulatingosmolarity to desirable levels for the symbiont, modulating pH todesirable levels for the symbiont, modulating secretions of organicacids, modulating the secretion of specific proteins, phenoliccompounds, nutrients, or the increased expression of a protein requiredfor host-biotic organisms interactions (e.g., a receptor, ligand, etc.).Additional mechanisms are known in the art and are encompassed by theinvention (see, for example, “Microbial Signalling and Communication”,eds., R. England, G. Hobbs, N. Bainton, and D. McL. Roberts, CambridgeUniversity Press, Cambridge, (1999); which is hereby incorporated hereinby reference).

In an alternative embodiment, a polynucleotide or polypeptide and/oragonist or antagonist of the present invention may decrease the hostorganisms ability to form biotic associations with another organism,either directly or indirectly. The mechanism by which a polynucleotideor polypeptide and/or agonist or antagonist of the present invention maydecrease the host organisms ability, either directly or indirectly, toinitiate and/or maintain biotic associations with another organism isvariable, though may include, modulating osmolarity to undesirablelevels, modulating pH to undesirable levels, modulating secretions oforganic acids, modulating the secretion of specific proteins, phenoliccompounds, nutrients, or the decreased expression of a protein requiredfor host-biotic organisms interactions (e.g., a receptor, ligand, etc.).Additional mechanisms are known in the art and are encompassed by theinvention (see, for example, “Microbial Signalling and Communication”,eds., R. England, G. Hobbs, N. Bainton, and D. McL. Roberts, CambridgeUniversity Press, Cambridge, (1999); which is hereby incorporated hereinby reference).

The hosts ability to maintain biotic associations with a particularpathogen has significant implications for the overall health and fitnessof the host. For example, human hosts have symbiosis with entericbacteria in their gastrointestinal tracts, particularly in the small andlarge intestine. In fact, bacteria counts in feces of the distal colonoften approach 10¹² per milliliter of feces. Examples of bowel flora inthe gastrointestinal tract are members of the Enterobacteriaceae,Bacteriodes, in addition to a-hemolytic streptococci, E. coli,Bifobacteria, Anaerobic cocci, Eubacteria, Costridia, lactobacilli, andyeasts. Such bacteria, among other things, assist the host in theassimilation of nutrients by breaking down food stuffs not typicallybroken down by the hosts digestive system, particularly in the hostsbowel. Therefore, increasing the hosts ability to maintain such a bioticassociation would help assure proper nutrition for the host.

Aberrations in the enteric bacterial population of mammals, particularlyhumans, has been associated with the following disorders: diarrhea,ileus, chronic inflammatory disease, bowel obstruction, duodenaldiverticula, biliary calculous disease, and malnutrition. Apolynucleotide or polypeptide and/or agonist or antagonist of thepresent invention are useful for treating, detecting, diagnosing,prognosing, and/or ameliorating, either directly or indirectly, and ofthe above mentioned diseases and/or disorders associated with aberrantenteric flora population.

The composition of the intestinal flora, for example, is based upon avariety of factors, which include, but are not limited to, the age,race, diet, malnutrition, gastric acidity, bile salt excretion, gutmotility, and immune mechanisms. As a result, the polynucleotides andpolypeptides, including agonists, antagonists, and fragments thereof,may modulate the ability of a host to form biotic associations byaffecting, directly or indirectly, at least one or more of thesefactors.

Although the predominate intestinal flora comprises anaerobic organisms,an underlying percentage represents aerobes (e.g., E. coli). This issignificant as such aerobes rapidly become the predominate organisms inintraabdominal infections—effectively becoming opportunistic early ininfection pathogenesis. As a result, there is an intrinsic need tocontrol aerobe populations, particularly for immune compromisedindividuals.

In a preferred embodiment, a polynucleotides and polypeptides, includingagonists, antagonists, and fragments thereof, are useful for inhibitingbiotic associations with specific enteric symbiont organisms in aneffort to control the population of such organisms.

Biotic associations occur not only in the gastrointestinal tract, butalso on an in the integument. As opposed to the gastrointestinal flora,the cutaneous flora is comprised almost equally with aerobic andanaerobic organisms. Examples of cutaneous flora are members of thegram-positive cocci (e.g., S. aureus, coagulase-negative staphylococci,micrococcus, M. sedentarius), gram-positive bacilli (e.g.,Corynebacterium species, C. minutissimum, Brevibacterium species,Propoionibacterium species, P. acnes), gram-negative bacilli (e.g.,Acinebacter species), and fungi (Pityrosporum orbiculare). Therelatively low number of flora associated with the integument is basedupon the inability of many organisms to adhere to the skin. Theorganisms referenced above have acquired this unique ability. Therefore,the polynucleotides and polypeptides of the present invention may haveuses which include modulating the population of the cutaneous flora,either directly or indirectly.

Aberrations in the cutaneous flora are associated with a number ofsignificant diseases and/or disorders, which include, but are notlimited to the following: impetigo, eethyma, blistering distaldactulitis, pustules, folliculitis, cutaneous abscesses, pittedkeratolysis, trichomycosis axcillaris, dermatophytosis complex, axillaryodor, erthyrasma, cheesy foot odor, acne, tinea versicolor, seborrheicdermititis, and Pityrosporum folliculitis, to name a few. Apolynucleotide or polypeptide and/or agonist or antagonist of thepresent invention are useful for treating, detecting, diagnosing,prognosing, and/or ameliorating, either directly or indirectly, and ofthe above mentioned diseases and/or disorders associated with aberrantcutaneous flora population.

Additional biotic associations, including diseases and disordersassociated with the aberrant growth of such associations, are known inthe art and are encompassed by the invention. See, for example,“Infectious Disease”, Second Edition, Eds., S. L., Gorbach, J. G.;Bartlett, and N. R., Blacklow, W.B. Saunders Company, Philadelphia,(1998); which is hereby incorporated herein by reference).

Pheromones

In another embodiment, a polynucleotide or polypeptide and/or agonist orantagonist of the present invention may increase the organisms abilityto synthesize and/or release a pheromone. Such a pheromone may, forexample, alter the organisms behavior and/or metabolism.

A polynucleotide or polypeptide and/or agonist or antagonist of thepresent invention may modulate the biosynthesis and/or release ofpheromones, the organisms ability to respond to pheromones (e.g.,behaviorally, and/or metabolically), and/or the organisms ability todetect pheromones. Preferably, any of the pheromones, and/or volatilesreleased from the organism, or induced, by a polynucleotide orpolypeptide and/or agonist or antagonist of the invention havebehavioral effects the organism.

Other Activities

The polypeptide of the present invention, as a result of the ability tostimulate vascular endothelial cell growth, may be employed in treatmentfor stimulating re-vascularization of ischemic tissues due to variousdisease conditions such as thrombosis, arteriosclerosis, and othercardiovascular conditions. These polypeptide may also be employed tostimulate angiogenesis and limb regeneration, as discussed above.

The polypeptide may also be employed for treating wounds due toinjuries, burns, post-operative tissue repair, and ulcers since they aremitogenic to various cells of different origins, such as fibroblastcells and skeletal muscle cells, and therefore, facilitate the repair orreplacement of damaged or diseased tissue.

The polypeptide of the present invention may also be employed stimulateneuronal growth and to treat, prevent, and/or diagnose neuronal damagewhich occurs in certain neuronal disorders or neuro-degenerativeconditions such as Alzheimer's disease, Parkinson's disease, andAIDS-related complex. The polypeptide of the invention may have theability to stimulate chondrocyte growth, therefore, they may be employedto enhance bone and periodontal regeneration and aid in tissuetransplants or bone grafts.

The polypeptide of the present invention may be also be employed toprevent skin aging due to sunburn by stimulating keratinocyte growth.

The polypeptide of the invention may also be employed for preventinghair loss, since FGF family members activate hair-forming cells andpromotes melanocyte growth. Along the same lines, the polypeptides ofthe present invention may be employed to stimulate growth anddifferentiation of hematopoietic cells and bone marrow cells when usedin combination with other cytokines.

The polypeptide of the invention may also be employed to maintain organsbefore transplantation or for supporting cell culture of primarytissues.

The polypeptide of the present invention may also be employed forinducing tissue of mesodermal origin to differentiate in early embryos.

The polypeptide or polynucleotides and/or agonist or antagonists of thepresent invention may also increase or decrease the differentiation orproliferation of embryonic stem cells, besides, as discussed above,hematopoietic lineage.

The polypeptide or polynucleotides and/or agonist or antagonists of thepresent invention may also be used to modulate mammaliancharacteristics, such as body height, weight, hair color, eye color,skin, percentage of adipose tissue, pigmentation, size, and shape (e.g.,cosmetic surgery). Similarly, polypeptides or polynucleotides and/oragonist or antagonists of the present invention may be used to modulatemammalian metabolism affecting catabolism, anabolism, processing,utilization, and storage of energy.

Polypeptide or polynucleotides and/or agonist or antagonists of thepresent invention may be used to change a mammal's mental state orphysical state by influencing biorhythms, caricadic rhythms, depression(including depressive diseases, disorders, and/or conditions), tendencyfor violence, tolerance for pain, reproductive capabilities (preferablyby Activin or Inhibin-like activity), hormonal or endocrine levels,appetite, libido, memory, stress, or other cognitive qualities.

Polypeptide or polynucleotides and/or agonist or antagonists of thepresent invention may also be used as a food additive or preservative,such as to increase or decrease storage capabilities, fat content,lipid, protein, carbohydrate, vitamins, minerals, cofactors or othernutritional components.

Polypeptide or polynucleotides and/or agonist or antagonists of thepresent invention may also be used to increase the efficacy of apharmaceutical composition, either directly or indirectly. Such a usemay be administered in simultaneous conjunction with saidpharmaceutical, or separately through either the same or different routeof administration (e.g., intravenous for the polynucleotide orpolypeptide of the present invention, and orally for the pharmaceutical,among others described herein.).

Polypeptide or polynucleotides and/or agonist or antagonists of thepresent invention may also be used to prepare individuals forextraterrestrial travel, low gravity environments, prolonged exposure toextraterrestrial radiation levels, low oxygen levels, reduction ofmetabolic activity, exposure to extraterrestrial pathogens, etc. Such ause may be administered either prior to an extraterrestrial event,during an extraterrestrial event, or both. Moreover, such a use mayresult in a number of beneficial changes in the recipient, such as, forexample, any one of the following, non-limiting, effects: an increasedlevel of hematopoietic cells, particularly red blood cells which wouldaid the recipient in coping with low oxygen levels; an increased levelof B-cells, T-cells, antigen presenting cells, and/or macrophages, whichwould aid the recipient in coping with exposure to extraterrestrialpathogens, for example; a temporary (i.e., reversible) inhibition ofhematopoietic cell production which would aid the recipient in copingwith exposure to extraterrestrial radiation levels; increase and/orstability of bone mass which would aid the recipient in coping with lowgravity environments; and/or decreased metabolism which wouldeffectively facilitate the recipients ability to prolong theirextraterrestrial travel by any one of the following, non-limiting means:(i) aid the recipient by decreasing their basal daily energyrequirements; (ii) effectively lower the level of oxidative and/ormetabolic stress in recipient (i.e., to enable recipient to cope withincreased extraterrestial radiation levels by decreasing the level ofinternal oxidative/metabolic damage acquired during normal basal energyrequirements; and/or (iii) enabling recipient to subsist at a lowermetabolic temperature (i.e., cryogenic, and/or sub-cryogenicenvironment).

Other Preferred Embodiments

Other preferred embodiments of the claimed invention include an isolatednucleic acid molecule comprising a nucleotide sequence which is at least95% identical to a sequence of at least about 50 contiguous nucleotidesin the nucleotide sequence of SEQ ID NO:X wherein X is any integer asdefined in Table I.

Also preferred is a nucleic acid molecule wherein said sequence ofcontiguous nucleotides is included in the nucleotide sequence of SEQ IDNO:X in the range of positions beginning with the nucleotide at aboutthe position of the “5′ NT of Start Codon of ORF” and ending with thenucleotide at about the position of the “3′ NT of ORF” as defined forSEQ ID NO:X in Table I.

Also preferred is an isolated nucleic acid molecule comprising anucleotide sequence which is at least 95% identical to a sequence of atleast about 150 contiguous nucleotides in the nucleotide sequence of SEQID NO:X.

Further preferred is an isolated nucleic acid molecule comprising anucleotide sequence which is at least 95% identical to a sequence of atleast about 500 contiguous nucleotides in the nucleotide sequence of SEQID NO:X.

A further preferred embodiment is a nucleic acid molecule comprising anucleotide sequence which is at least 95% identical to the nucleotidesequence of SEQ ID NO:X beginning with the nucleotide at about theposition of the “5′ NT of ORF” and ending with the nucleotide at aboutthe position of the “3′ NT of ORF” as defined for SEQ ID NO:X in TableI.

A further preferred embodiment is an isolated nucleic acid moleculecomprising a nucleotide sequence which is at least 95% identical to thecomplete nucleotide sequence of SEQ ID NO:X.

Also preferred is an isolated nucleic acid molecule which hybridizesunder stringent hybridization conditions to a nucleic acid molecule,wherein said nucleic acid molecule which hybridizes does not hybridizeunder stringent hybridization conditions to a nucleic acid moleculehaving a nucleotide sequence consisting of only A residues or of only Tresidues.

Also preferred is a composition of matter comprising a DNA moleculewhich comprises a cDNA clone identified by a cDNA Clone Identifier inTable I, which DNA molecule is contained in the material deposited withthe American Type Culture Collection and given the ATCC Deposit Numbershown in Table I for said cDNA Clone Identifier.

Also preferred is an isolated nucleic acid molecule comprising anucleotide sequence which is at least 95% identical to a sequence of atleast 50 contiguous nucleotides in the nucleotide sequence of a cDNAclone identified by a cDNA Clone Identifier in Table I, which DNAmolecule is contained in the deposit given the ATCC Deposit Number shownin Table I.

Also preferred is an isolated nucleic acid molecule, wherein saidsequence of at least 50 contiguous nucleotides is included in thenucleotide sequence of the complete open reading frame sequence encodedby said cDNA clone.

Also preferred is an isolated nucleic acid molecule comprising anucleotide sequence which is at least 95% identical to sequence of atleast 150 contiguous nucleotides in the nucleotide sequence encoded bysaid cDNA clone.

A further preferred embodiment is an isolated nucleic acid moleculecomprising a nucleotide sequence which is at least 95% identical tosequence of at least 500 contiguous nucleotides in the nucleotidesequence encoded by said cDNA clone.

A further preferred embodiment is an isolated nucleic acid moleculecomprising a nucleotide sequence which is at least 95% identical to thecomplete nucleotide sequence encoded by said cDNA clone.

A further preferred embodiment is a method for detecting in a biologicalsample a nucleic acid molecule comprising a nucleotide sequence which isat least 95% identical to a sequence of at least 50 contiguousnucleotides in a sequence selected from the group consisting of: anucleotide sequence of SEQ ID NO:X wherein X is any integer as definedin Table I; and a nucleotide sequence encoded by a cDNA clone identifiedby a cDNA Clone Identifier in Table I and contained in the deposit withthe ATCC Deposit Number shown for said cDNA clone in Table I; whichmethod comprises a step of comparing a nucleotide sequence of at leastone nucleic acid molecule in said sample with a sequence selected fromsaid group and determining whether the sequence of said nucleic acidmolecule in said sample is at least 95% identical to said selectedsequence.

Also preferred is the above method wherein said step of comparingsequences comprises determining the extent of nucleic acid hybridizationbetween nucleic acid molecules in said sample and a nucleic acidmolecule comprising said sequence selected from said group. Similarly,also preferred is the above method wherein said step of comparingsequences is performed by comparing the nucleotide sequence determinedfrom a nucleic acid molecule in said sample with said sequence selectedfrom said group. The nucleic acid molecules can comprise DNA moleculesor RNA molecules.

A further preferred embodiment is a method for identifying the species,tissue or cell type of a biological sample which method comprises a stepof detecting nucleic acid molecules in said sample, if any, comprising anucleotide sequence that is at least 95% identical to a sequence of atleast 50 contiguous nucleotides in a sequence selected from the groupconsisting of: a nucleotide sequence of SEQ ID NO:X wherein X is anyinteger as defined in Table I; and a nucleotide sequence encoded by acDNA clone identified by a cDNA Clone Identifier in Table I andcontained in the deposit with the ATCC Deposit Number shown for saidcDNA clone in Table I.

The method for identifying the species, tissue or cell type of abiological sample can comprise a step of detecting nucleic acidmolecules comprising a nucleotide sequence in a panel of at least twonucleotide sequences, wherein at least one sequence in said panel is atleast 95% identical to a sequence of at least 50 contiguous nucleotidesin a sequence selected from said group.

Also preferred is a method for diagnosing in a subject a pathologicalcondition associated with abnormal structure or expression of a geneencoding a protein identified in Table I, which method comprises a stepof detecting in a biological sample obtained from said subject nucleicacid molecules, if any, comprising a nucleotide sequence that is atleast 95% identical to a sequence of at least 50 contiguous nucleotidesin a sequence selected from the group consisting of: a nucleotidesequence of SEQ ID NO:X wherein X is any integer as defined in Table I;and a nucleotide sequence encoded by a cDNA clone identified by a cDNAClone Identifier in Table I and contained in the deposit with the ATCCDeposit Number shown for said cDNA clone in Table I.

The method for diagnosing a pathological condition can comprise a stepof detecting nucleic acid molecules comprising a nucleotide sequence ina panel of at least two nucleotide sequences, wherein at least onesequence in said panel is at least 95% identical to a sequence of atleast 50 contiguous nucleotides in a sequence selected from said group.

Also preferred is a composition of matter comprising isolated nucleicacid molecules wherein the nucleotide sequences of said nucleic acidmolecules comprise a panel of at least two nucleotide sequences, whereinat least one sequence in said panel is at least 95% identical to asequence of at least 50 contiguous nucleotides in a sequence selectedfrom the group consisting of: a nucleotide sequence of SEQ ID NO:Xwherein X is any integer as defined in Table I; and a nucleotidesequence encoded by a cDNA clone identified by a cDNA Clone Identifierin Table I and contained in the deposit with the ATCC Deposit Numbershown for said cDNA clone in Table I. The nucleic acid molecules cancomprise DNA molecules or RNA molecules.

Also preferred is an isolated polypeptide comprising an amino acidsequence at least 90% identical to a sequence of at least about 10contiguous amino acids in the amino acid sequence of SEQ ID NO:Y whereinY is any integer as defined in Table I.

Also preferred is a polypeptide, wherein said sequence of contiguousamino acids is included in the amino acid sequence of SEQ ID NO:Y in therange of positions “Total AA of the Open Reading Frame (ORF)” as setforth for SEQ ID NO:Y in Table I.

Also preferred is an isolated polypeptide comprising an amino acidsequence at least 95% identical to a sequence of at least about 30contiguous amino acids in the amino acid sequence of SEQ ID NO:Y.

Further preferred is an isolated polypeptide comprising an amino acidsequence at least 95% identical to a sequence of at least about 100contiguous amino acids in the amino acid sequence of SEQ ID NO:Y.

Further preferred is an isolated polypeptide comprising an amino acidsequence at least 95% identical to the complete amino acid sequence ofSEQ ID NO:Y.

Further preferred is an isolated polypeptide comprising an amino acidsequence at least 90% identical to a sequence of at least about 10contiguous amino acids in the complete amino acid sequence of a proteinencoded by a cDNA clone identified by a cDNA Clone Identifier in Table Iand contained in the deposit with the ATCC Deposit Number shown for saidcDNA clone in Table I.

Also preferred is a polypeptide wherein said sequence of contiguousamino acids is included in the amino acid sequence of the proteinencoded by a cDNA clone identified by a cDNA Clone Identifier in Table Iand contained in the deposit with the ATCC Deposit Number shown for saidcDNA clone in Table I.

Also preferred is an isolated polypeptide comprising an amino acidsequence at least 95% identical to a sequence of at least about 30contiguous amino acids in the amino acid sequence of the protein encodedby a cDNA clone identified by a cDNA Clone Identifier in Table I andcontained in the deposit with the ATCC Deposit Number shown for saidcDNA clone in Table I.

Also preferred is an isolated polypeptide comprising an amino acidsequence at least 95% identical to a sequence of at least about 100contiguous amino acids in the amino acid sequence of the protein encodedby a cDNA clone identified by a cDNA Clone Identifier in Table I andcontained in the deposit with the ATCC Deposit Number shown for saidcDNA clone in Table I.

Also preferred is an isolated polypeptide comprising an amino acidsequence at least 95% identical to the amino acid sequence of theprotein encoded by a cDNA clone identified by a cDNA Clone Identifier inTable I and contained in the deposit with the ATCC Deposit Number shownfor said cDNA clone in Table I.

Further preferred is an isolated antibody which binds specifically to apolypeptide comprising an amino acid sequence that is at least 90%identical to a sequence of at least 10 contiguous amino acids in asequence selected from the group consisting of: an amino acid sequenceof SEQ ID NO:Y wherein Y is any integer as defined in Table I; and acomplete amino acid sequence of a protein encoded by a cDNA cloneidentified by a cDNA Clone Identifier in Table I and contained in thedeposit with the ATCC Deposit Number shown for said cDNA clone in TableI.

Further preferred is a method for detecting in a biological sample apolypeptide comprising an amino acid sequence which is at least 90%identical to a sequence of at least 10 contiguous amino acids in asequence selected from the group consisting of: an amino acid sequenceof SEQ ID NO:Y wherein Y is any integer as defined in Table I; and acomplete amino acid sequence of a protein encoded by a cDNA cloneidentified by a cDNA Clone Identifier in Table I and contained in thedeposit with the ATCC Deposit Number shown for said cDNA clone in TableI; which method comprises a step of comparing an amino acid sequence ofat least one polypeptide molecule in said sample with a sequenceselected from said group and determining whether the sequence of saidpolypeptide molecule in said sample is at least 90% identical to saidsequence of at least 10 contiguous amino acids.

Also preferred is the above method wherein said step of comparing anamino acid sequence of at least one polypeptide molecule in said samplewith a sequence selected from said group comprises determining theextent of specific binding of polypeptides in said sample to an antibodywhich binds specifically to a polypeptide comprising an amino acidsequence that is at least 90% identical to a sequence of at least 10contiguous amino acids in a sequence selected from the group consistingof: an amino acid sequence of SEQ ID NO:Y wherein Y is any integer asdefined in Table I; and a complete amino acid sequence of a proteinencoded by a cDNA clone identified by a cDNA Clone Identifier in Table Iand contained in the deposit with the ATCC Deposit Number shown for saidcDNA clone in Table I.

Also preferred is the above method wherein said step of comparingsequences is performed by comparing the amino acid sequence determinedfrom a polypeptide molecule in said sample with said sequence selectedfrom said group.

Also preferred is a method for identifying the species, tissue or celltype of a biological sample which method comprises a step of detectingpolypeptide molecules in said sample, if any, comprising an amino acidsequence that is at least 90% identical to a sequence of at least 10contiguous amino acids in a sequence selected from the group consistingof: an amino acid sequence of SEQ ID NO:Y wherein Y is any integer asdefined in Table I; and a complete amino acid sequence of a proteinencoded by a cDNA clone identified by a cDNA Clone Identifier in Table Iand contained in the deposit with the ATCC Deposit Number shown for saidcDNA clone in Table I.

Also preferred is the above method for identifying the species, tissueor cell type of a biological sample, which method comprises a step ofdetecting polypeptide molecules comprising an amino acid sequence in apanel of at least two amino acid sequences, wherein at least onesequence in said panel is at least 90% identical to a sequence of atleast 10 contiguous amino acids in a sequence selected from the abovegroup.

Also preferred is a method for diagnosing a pathological conditionassociated with an organism with abnormal structure or expression of agene encoding a protein identified in Table I, which method comprises astep of detecting in a biological sample obtained from said subjectpolypeptide molecules comprising an amino acid sequence in a panel of atleast two amino acid sequences, wherein at least one sequence in saidpanel is at least 90% identical to a sequence of at least 10 contiguousamino acids in a sequence selected from the group consisting of: anamino acid sequence of SEQ ID NO:Y wherein Y is any integer as definedin Table I; and a complete amino acid sequence of a protein encoded by acDNA clone identified by a cDNA Clone Identifier in Table I andcontained in the deposit with the ATCC Deposit Number shown for saidcDNA clone in Table I.

In any of these methods, the step of detecting said polypeptidemolecules includes using an antibody.

Also preferred is an isolated nucleic acid molecule comprising anucleotide sequence which is at least 95% identical to a nucleotidesequence encoding a polypeptide wherein said polypeptide comprises anamino acid sequence that is at least 90% identical to a sequence of atleast 10 contiguous amino acids in a sequence selected from the groupconsisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is anyinteger as defined in Table I; and a complete amino acid sequence of aprotein encoded by a cDNA clone identified by a cDNA Clone Identifier inTable I and contained in the deposit with the ATCC Deposit Number shownfor said cDNA clone in Table I.

Also preferred is an isolated nucleic acid molecule, wherein saidnucleotide sequence encoding a polypeptide has been optimized forexpression of said polypeptide in a prokaryotic host.

Also preferred is an isolated nucleic acid molecule, wherein saidpolypeptide comprises an amino acid sequence selected from the groupconsisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is anyinteger as defined in Table I; and a complete amino acid sequence of aprotein encoded by a cDNA clone identified by a cDNA Clone Identifier inTable I and contained in the deposit with the ATCC Deposit Number shownfor said cDNA clone in Table I.

Further preferred is a method of making a recombinant vector comprisinginserting any of the above isolated nucleic acid molecule(s) into avector. Also preferred is the recombinant vector produced by thismethod. Also preferred is a method of making a recombinant host cellcomprising introducing the vector into a host cell, as well as therecombinant host cell produced by this method.

Also preferred is a method of making an isolated polypeptide comprisingculturing this recombinant host cell under conditions such that saidpolypeptide is expressed and recovering said polypeptide. Also preferredis this method of making an isolated polypeptide, wherein saidrecombinant host cell is a eukaryotic cell and said polypeptide is aprotein comprising an amino acid sequence selected from the groupconsisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is aninteger set forth in Table I and said position of the “Total AA of ORF”of SEQ ID NO:Y is defined in Table I; and an amino acid sequence of aprotein encoded by a cDNA clone identified by a cDNA Clone Identifier inTable I and contained in the deposit with the ATCC Deposit Number shownfor said cDNA clone in Table I. The isolated polypeptide produced bythis method is also preferred.

Also preferred is a method of treatment of an individual in need of anincreased level of a protein activity, which method comprisesadministering to such an individual a pharmaceutical compositioncomprising an amount of an isolated polypeptide, polynucleotide, orantibody of the claimed invention effective to increase the level ofsaid protein activity in said individual.

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as limiting.

REFERENCES

-   Altschul, S. F., T. L. Madden, et al. (1997). “Gapped BLAST and    PSI-BLAST: a new generation of protein database search programs.”    Nucleic Acids Res 25 (17): 3389-402.-   Bateman, A., E. Birney, et al. (2000). Nucleic Acids Res 28(1):    263-6.-   Burge, C. and S. Karlin (1997)., J Mol Biol 268(1): 78-94.-   Fauman, E. B. and M. A. Saper (1996)., Trends Biochem Sci 21(11):    413-7.-   Sonnhammer, E. L., S. R. Eddy, et al. (1997), Proteins 28(3):    405-20.-   Bernstein, F C, Koetzle, T F, Williams, G J B, Meyer, E F Jr.,    Brice, M D, Rodgers, JR, Kennard, O, Simanouchi, T, Tasumi, M. 1977.    The Protein Data Bank: A computer-based archival file for    macromolecular structures. J. Mol. Biol. 112:535-542.-   Bohm H-J, LUDI: rule-based automatic design of new substituents for    enzyme inhibitor leads. J. Comp. Aid. Molec. Design 6:61-78 (1992)-   Cabral, J. H. M., Lee, A., Cardozo T; Totrov M; Abagyan R Homology    modeling by the ICM method. Proteins 23, 403-14 (1995).-   Cardozo, T., Totrov, M., Abagyan, R. Homology modeling by the ICM    method. Proteins 23:403-14, 1995.-   Fauman, E. and Saper, M. Structure and function of the protein    tyrosine phosphatases. Trends Biochem. Sci. 21:413-7 (1996).-   Goodford, P. J. A computational procedure for determining    energetically favorable binding sites on biologically important    macromolecules. J. Med. Chem. 28:849-857 (1985)-   Goodsell, D. S. and Olsen, A. J. Automated docking of substrates to    proteins by simulated annealing. Proteins 8:195-202 (1990)-   Greer J Comparative modeling of homologous proteins. Meth. Enzymol.    202:239-52 (1991).-   Hendlich M; Lackner P; Weitckus S; Floeckner H; Froschauer R;    Gottsbacher K; Casari G; Sippi M J Identification of native protein    folds amongst a large number of incorrect models. The calculation of    low energy conformations from potentials of mean force. J. Mol.    Biol. 216, 167-80 (1990).-   Jia, Z., Badford, D., Flint, A. J., and Tonks, N. K. Structural    basis for phosphotyrosine peptide recognition by protein tyrosine    phosphatase 1B. Science 268:1754-8, 1995.-   Kuntz I D, Blaney J M, Oatley S J, Langridge R, Ferrin T E. A    geometric approach to macromolecule-ligand interactions. J. Mol.    Biol. 161:269-288 (1982)-   Lesk, A. M., Boswell, D. R., Homology Modeling: Inferences from    Tables of Aligned Sequences. Curr. Op. Struc. Biol. 2: 242-247    (1992)-   Levitt, M. Accurate modeling of protein conformation by automatic    segment matching. J Mol Biol 226:507-33 (1992)-   Martin, Y. C. 3D database searching in drug design. J. Med. Chem.    35:2145-2154 (1992)-   Novotny J; Rashin A A; Bruccoleri R E. Criteria that discriminate    between native proteins and incorrectly folded models. Proteins,    4:19-30 (1988).-   Pearson W R Rapid and sensitive sequence comparison with FASTP and    FASTA. Methods In Enzymology 18363-98 (1990).-   Pearson, W. R. Rapid and sensitive sequence comparison with FASTP    and FASTA. Meth. Enzymol. 183:63-98, 1990.-   Sali A; Potterton L; Yuan F; van Vlijmen H; Karplus M Evaluation of    comparative protein modeling by MODELLER. Proteins 23:318-26 (1995).-   Stewart, A. E., Dowd, S., Keyse, S. M. and McDonald, N. Q. Crystal    structure of the MAPK phosphatase Pyst1 catalytic domain and    implications for regulated activation. Nat. Struct. Biol. 6:174-80    (1999).-   Yuvaniyama, J.; Denu, J. M.; Dixon, J. E. and Saper, M. A. Crystal    structure of the dual specificity protein phosphatase vhr. Science    272:1328-31 (1996).

EXAMPLES Description of the Preferred Embodiments Example 1 Method ofIdentifying the Novel BMY_HPP Human Phosphatases of the PresentInvention

Polynucleotide sequences encoding the novel BMY_HPP phosphoproteinphosphatases of the present invention were identified by a combinationof the following methods:

Homology-based searches using the TBLASTN program [Altschul, 1997] tocompare known phosphoprotein phosphatases with human genomic (gDNA) andEST sequences. EST or gDNA sequences having significant homology to oneor more of the known phosphatases listed in Table III (expect score lessthan or equal to 1×10⁻³) were retained for further analysis.

Hidden Markov Model (HMM) searches using PFAM motifs (listed in TableIV) [Bateman, 2000 #9; Sonnhammer, 1997] were used to search humangenomic sequence using the Genewise program. EST or gDNA sequenceshaving a significant score (greater than or equal to 10) with any of thefollowing motifs were retained for further analysis.

HMM searches using PFAM motifs (listed in Table IV) were used to searchpredicted protein sequences identified by GENSCAN analysis of humangenomic sequence [Burge, 1997 #10]. gDNA sequences having a significantscore (greater than or equal to 10) with any of the following motifswere retained for further analysis.

TABLE IV PFAM motifs used to identify phopsphoprotein phosphatases PFAMMotif Name Accession No. Description DSPc PF00782 Dual specificityphosphatase, catalytic domain ST_phosphatase PF00149 Ser/Thr proteinphosphatase Y_phosphatase PF00102 Protein-tyrosine phosphatase

Once a bacterial artificial chromosomes (BACs) encoding a novelphosphoprotein phosphatase was identified by any one of the methodsabove, additional potential exons were identified using GENSCAN analysisof all nearby BACs (identified by the Golden Path tiling map, UCSC).Intron/exon boundaries, transcript cDNA sequence and protein sequencewere determined using GENSCAN. The predicted protein sequence were usedto identify the most closely related known phosphatase using the BLASTPprogram as described in herein.

In the case of BMY_HPP5, BMY_HPP5 was identified as an Incyte EST (ID4155374) with homology to known protein phosphatases and significantexpression in the central nervous system. The Incyte clone sequence wasused to design oligonucleotides for isolation of additional cDNAs. SuchcDNAs have been recovered and sequenced and compared to a full-lengthIncyte template (assembly of EST sequences) (ID 1026659.7). The BMY_HPP5cDNA has significant identity to Incyte 1026659.7 but diverges at thefive-prime and three-prime ends, suggesting that it may be analternatively spliced product of the same gene.

Example 2 Cloning of the Novel Human BMY_HPP Phosphatases of the PresentInvention

A variety of methods known in the art may be used for cloning the novelBMY_HPP phosphatases of the present invention. Breifly, using thepredicted or observed cDNA sequences for the BMY-HPP genes of thepresent invention, antisense oligonucleotides with biotin on the 5′ endcould be designed (the sequences of these oligos are provided in TableVI). These oligos will be used to isolate cDNA clones according to thefollowing procedure:

One microliter (one hundred and fifty nanograms) of a biotinylated oligois added to six microliters (six micrograms) of a mixture ofsingle-stranded covalently closed circular cDNA libraries (suchlibraries are commercially available from Life Technologies, Rockville,Md., or may be created using routine methods known in the art) and sevenmicroliters of 100% formamide in a 0.5 ml PCR tube. The cDNA librariesused for specific BMY_HPP genes will be determined by the results of theexpression patterns as described herein.

The mixture is heated in a thermal cycler to 95° C. for 2 mins.

Fourteen microliters of 2× hybridization buffer (50% formamide, 1.5 MNaCl, 0.04 M NaPO₄, pH 7.2, 5 mM EDTA, 0.2% SDS) wis added to the heatedprobe/cDNA library mixture and incubated at 42° C. for 26 hours.

Hybrids between the biotinylated oligo and the circular cDNA areisolated by diluting the hybridization mixture to 220 microliters in asolution containing 1 M NaCl, 10 mM Tris-HCl pH 7.5, 1 mM EDTA, pH 8.0and adding 125 microliters of streptavidin magnetic beads. This solutionis incubated at 42° C. for 60 mins, mixing every 5 mins to resuspend thebeads.

The beads are separated from the solution with a magnet and the beadswashed three times in 200 microliters of 0.1×SSPE, 0.1% SDS at 45° C.

The single stranded cDNAs are released from the biotinlyatedoligo/streptavidin magnetic bead complex by adding 50 microliters of 0.1N NaOH and incubating at room temperature for 10 mins.

The cDNAs are precipitated by adding six microliters of 3 M SodiumAcetate, 5 micrograms of glycogen and 120 microliters of 100% ethanolfollowed by centrifugation.

The cDNAs are resuspended in 12 microliters of TE (10 mM Tris-HCl, pH8.0), 1 mM EDTA, pH 8.0).

The single stranded cDNAs are converted into double stranded moleculesin a thermal cycler by mixing 5 microliters of the captured DNA with 1.5microliters of a standard SP6 primer (homologous to a sequence on thecDNA cloning vector) at 10 micromolar concentration and 1.5 microlitersof 10×PCR buffer. The mixture is heated to 95° C. for 20 seconds, thenramped down to 59° C. At this time 15 microliters of a repair mixpreheated to 70° C. is added (repair mix contains 4 microliters of 5 mMdNTPs (1.25 mM each), 1.5 microliters of 10×PCR buffer, 9.25 microlitersof water, and 0.25 microliters of Taq polymerase). The solution isramped back to 73° C. and incubated for 23 mins.

The repaired DNA was precipitated as described above and resuspended in10 microliters of TE.

Two microliters of double-stranded cDNA are used to transform E. coliDH12S cells by electroporation.

The resulting colonies are screened by PCR, using a primer pair designedto identify the proper cDNAs (primer sequences, as provided in Table VI,may be used).

Those cDNA clones that are positive by PCR are then assessed todetermine the inserts size. Two clones for each BMY_HPP gene are chosenfor DNA sequencing using standard methods known in the art and describedherein.

The polynucleotide(s) of the present invention, the polynucleotideencoding the polypeptide of the present invention, or the polypeptideencoded by the deposited clone may represent partial, or incompleteversions of the complete coding region (i.e., full-length gene). Severalmethods are known in the art for the identification of the 5′ or 3′non-coding and/or coding portions of a gene which may not be present inthe deposited clone. The methods that follow are exemplary and shouldnot be construed as limiting the scope of the invention. These methodsinclude but are not limited to, filter probing, clone enrichment usingspecific probes, and protocols similar or identical to 5′ and 3′ “RACE”protocols that are well known in the art. For instance, a method similarto 5′ RACE is available for generating the missing 5′ end of a desiredfull-length transcript. (Fromont-Racine et al., Nucleic Acids Res.21(7):1683-1684 (1993)).

Briefly, a specific RNA oligonucleotide is ligated to the 5′ ends of apopulation of RNA presumably containing full-length gene RNAtranscripts. A primer set containing a primer specific to the ligatedRNA oligonucleotide and a primer specific to a known sequence of thegene of interest is used to PCR amplify the 5′ portion of the desiredfull-length gene. This amplified product may then be sequenced and usedto generate the full-length gene.

This above method starts with total RNA isolated from the desiredsource, although poly-A+ RNA can be used. The RNA preparation can thenbe treated with phosphatase if necessary to eliminate 5′ phosphategroups on degraded or damaged RNA that may interfere with the later RNAligase step. The phosphatase should then be inactivated and the RNAtreated with tobacco acid pyrophosphatase in order to remove the capstructure present at the 5′ ends of messenger RNAs. This reaction leavesa 5′ phosphate group at the 5′ end of the cap cleaved RNA which can thenbe ligated to an RNA oligonucleotide using T4 RNA ligase.

This modified RNA preparation is used as a template for first strandcDNA synthesis using a gene specific oligonucleotide. The first strandsynthesis reaction is used as a template for PCR amplification of thedesired 5′ end using a primer specific to the ligated RNAoligonucleotide and a primer specific to the known sequence of the geneof interest. The resultant product is then sequenced and analyzed toconfirm that the 5′ end sequence belongs to the desired gene. Moreover,it may be advantageous to optimize the RACE protocol to increase theprobability of isolating additional 5′ or 3′ coding or non-codingsequences. Various methods of optimizing a RACE protocol are known inthe art, though a detailed description summarizing these methods can befound in B. C. Schaefer, Anal. Biochem., 227:255-273, (1995).

An alternative method for carrying out 5′ or 3′ RACE for theidentification of coding or non-coding sequences is provided by Frohman,M. A., et al., Proc. Nat'l. Acad. Sci. USA, 85:8998-9002 (1988).Briefly, a cDNA clone missing either the 5′ or 3′ end can bereconstructed to include the absent base pairs extending to thetranslational start or stop codon, respectively. In some cases, cDNAsare missing the start of translation, therefor. The following brieflydescribes a modification of this original 5′ RACE procedure. Poly A+ ortotal RNAs reverse transcribed with Superscript II (Gibco/BRL) and anantisense or I complementary primer specific to the cDNA sequence. Theprimer is removed from the reaction with a Microcon Concentrator(Amicon). The first-strand cDNA is then tailed with dATP and terminaldeoxynucleotide transferase (Gibco/BRL). Thus, an anchor sequence isproduced which is needed for PCR amplification. The second strand issynthesized from the dA-tail in PCR buffer, Taq DNA polymerase(Perkin-Elmer Cetus), an oligo-dT primer containing three adjacentrestriction sites (XhoIJ Sail and ClaI) at the 5′ end and a primercontaining just these restriction sites. This double-stranded cDNA isPCR amplified for 40 cycles with the same primers as well as a nestedcDNA-specific antisense primer. The PCR products are size-separated onan ethidium bromide-agarose gel and the region of gel containing cDNAproducts the predicted size of missing protein-coding DNA is removed.cDNA is purified from the agarose with the Magic PCR Prep kit (Promega),restriction digested with XhoI or SalI, and ligated to a plasmid such aspBluescript SKII (Stratagene) at XhoI and EcoRV sites. This DNA istransformed into bacteria and the plasmid clones sequenced to identifythe correct protein-coding inserts. Correct 5′ ends are confirmed bycomparing this sequence with the putatively identified homologue andoverlap with the partial cDNA clone. Similar methods known in the artand/or commercial kits are used to amplify and recover 3′ ends.

Several quality-controlled kits are commercially available for purchase.Similar reagents and methods to those above are supplied in kit formfrom Gibco/BRL for both 5′ and 3′ RACE for recovery of full lengthgenes. A second kit is available from Clontech which is a modificationof a related technique, SLIC (single-stranded ligation tosingle-stranded cDNA), developed by Dumas et al., Nucleic Acids Res.,19:5227-32 (1991). The major differences in procedure are that the RNAis alkaline hydrolyzed after reverse transcription and RNA ligase isused to join a restriction site-containing anchor primer to thefirst-strand cDNA. This obviates the necessity for the dA-tailingreaction which results in a polyT stretch that is difficult to sequencepast.

An alternative to generating 5′ or 3′ cDNA from RNA is to use cDNAlibrary double-stranded DNA. An asymmetric PCR-amplified antisense cDNAstrand is synthesized with an antisense cDNA-specific primer and aplasmid-anchored primer. These primers are removed and a symmetric PCRreaction is performed with a nested cDNA-specific antisense primer andthe plasmid-anchored primer.

RNA Ligase Protocol for Generating the 5′ or 3′ End Sequences to ObtainFull Length Genes

Once a gene of interest is identified, several methods are available forthe identification of the 5′ or 3′ portions of the gene which may not bepresent in the original cDNA plasmid. These methods include, but are notlimited to, filter probing, clone enrichment using specific probes andprotocols similar and identical to 5′ and 3′RACE. While the full-lengthgene may be present in the library and can be identified by probing, auseful method for generating the 5′ or 3′ end is to use the existingsequence information from the original cDNA to generate the missinginformation. A method similar to 5′RACE is available for generating themissing 5′ end of a desired full-length gene. (This method was publishedby Fromont-Racine et al., Nucleic Acids Res., 21(7): 1683-1684 (1993)).Briefly, a specific RNA oligonucleotide is ligated to the 5′ ends of apopulation of RNA presumably 30 containing full-length gene RNAtranscript and a primer set containing a primer specific to the ligatedRNA oligonucleotide and a primer specific to a known sequence of thegene of interest, is used to PCR amplify the 5′ portion of the desiredfull length gene which may then be sequenced and used to generate thefull length gene. This method starts with total RNA isolated from thedesired source, poly A RNA may be used but is not a prerequisite forthis procedure. The RNA preparation may then be treated with phosphataseif necessary to eliminate 5′ phosphate groups on degraded or damaged RNAwhich may interfere with the later RNA ligase step. The phosphatase ifused is then inactivated and the RNA is treated with tobacco acidpyrophosphatase in order to remove the cap structure present at the 5′ends of messenger RNAs. This reaction leaves a 5′ phosphate group at the5′ end of the cap cleaved RNA which can then be ligated to an RNAoligonucleotide using T4 RNA ligase. This modified RNA preparation canthen be used as a template for first strand cDNA synthesis using a genespecific oligonucleotide. The first strand synthesis reaction can thenbe used as a template for PCR amplification of the desired 5′ end usinga primer specific to the ligated RNA oligonucleotide and a primerspecific to the known sequence of interest. The resultant product isthen sequenced and analyzed to confirm that the 5′ end sequence belongsto the relevant family.

Representative primers for cloning any one of the human phosphatases ofthe present invention are provided in Table VI herein as ‘Left CloningPrimer’, ‘Right Cloning Primer’, ‘Internal RevComp Cloning Primer’,and/or ‘Internal Cloning Primer’. Other primers could be subsititutedfor any of the above as would be appreciated by one skilled in the art.

In the case of the full-length BMY_HPP1, BMY_HPP1 was cloned using thepolynucleotide sequences of the identified BMY_HPP1 fragments BMY_HPP1_A(SEQ ID NO:1) and BMY_HPP1_B (SEQ ID NO:3) to design the followingantisense 80 bp oligo with biotin on the 5′ end:

Name Sequence Phos4-80b 5′bTGACAATGGATAGCTACTTTTCCTTCCTGTAAGGCAAATGTCATCACCTTCACCATATCTAGGATAGTAGTAAGAG ACGC -3 (SEQ ID NO:45)

One microliter (one hundred and fifty nanograms) of the gel-purifiedbiotinylated PCR fragment was added to six microliters (six micrograms)of a single-stranded covalently closed circular brain, fetal brain, bonemarrow, prostate, spleen, testis, and thymus cDNA libraries and sevenmicroliters of 100% formamide in a 0.5 ml PCR tube. The mixture washeated in a thermal cycler to 95° C. for 2 mins. Fourteen microliters of2× hybridization buffer (50% formamide, 1.5 M NaCl, 0.04 M NaPO₄, pH7.2, 5 mM EDTA, 0.2% SDS) was added to the heated probe/cDNA librarymixture and incubated at 42° C. for 26 hours. Hybrids between thebiotinylated oligo and the circular cDNA were isolated by diluting thehybridization mixture to 220 microliters in a solution containing 1 MNaCl, 10 mM Tris-HCl pH 7.5, 1 mM EDTA, pH 8.0 and adding 125microliters of streptavidin magnetic beads. This solution was incubatedat 42° C. for 60 mins, mixing every 5 mins to resuspend the beads. Thebeads were separated from the solution with a magnet and the beadswashed three times in 200 microliters of 0.1×SSPE, 0.1% SDS at 45° C.

The single stranded cDNAs were released from the biotinlyatedprobe/streptavidin magnetic bead complex by adding 50 microliters of 0.1N NaOH and incubating at room temperature for 10 mins. Six microlitersof 3 M Sodium Acetate was added along with 15 micrograms of glycogen andthe solution ethanol precipitated with 120 microliters of 100% ethanol.The DNA was resuspend in 12 microliters of TE (10 mM Tris-HCl, pH 8.0),1 mM EDTA, pH 8.0). The single stranded cDNA was converted into doublestrands in a thermal cycler by mixing 5 microliters of the captured DNAwith 1.5 microliters 10 micromolar standard SP6 primer (homologous to asequence on the cDNA cloning vector) and 1.5 microliters of 10×PCRbuffer. The mixture was heated to 95° C. for 20 seconds, then rampeddown to 59° C. At this time 15 microliters of a repair mix, that waspreheated to 70° C. (Repair mix contains 4 microliters of 5 mM dNTPs(1.25 mM each), 1.5 microliters of 10×PCR buffer, 9.25 microliters ofwater, and 0.25 microliters of Taq polymerase). The solution was rampedback to 73° C. and incubated for 23 mins. The repaired DNA was ethanolprecipitated and resuspended in 10 microliters of TE. Two microliterswere electroporated in E. coli DH12S cells and resulting colonies werescreened by PCR, using the following primer pair number:

Name Sequence Phos2-2s TACAATTTCGGATGGAAGGATTAT (SEQ ID NO:154) Phos2-2aGCATGACAATGGATAGCTACTTT (SEQ ID NO: 155)

The sequence of the BMY_HPP1 polynucleotide was sequenced and isprovided in FIGS. 20A-D (SEQ ID NO:149).

In the case of the full-length BMY_HPP2, BMY_HPP1 was cloned using thepolynucleotide sequences of the identified BMY_HPP2 fragment (SEQ IDNO:5) to design the following antisense 80 bp oligo with biotin on the5′ end:

Name Sequence Phos2-80b 5′bGTGCCGCACGCCCAGGTCCAACAGGAACTGGTAGTGGGCGGGGAGCCGCGGCAGCGCCAGTCCCGCCAGCCGG CCCGGA -3 (SEQ ID NO:51)

One microliter (one hundred and fifty nanograms) of the gel-purifiedbiotinylated PCR fragment was added to six microliters (six micrograms)of a single-stranded covalently closed circular brain, fetal brain, bonemarrow, prostate, spleen, testis, and thymus cDNA libraries and sevenmicroliters of 100% formamide in a 0.5 ml PCR tube. The mixture washeated in a thermal cycler to 95° C. for 2 mins. Fourteen microliters of2× hybridization buffer (50% formamide, 1.5 M NaCl, 0.04 M NaPO₄, pH7.2, 5 mM EDTA, 0.2% SDS) was added to the heated probe/cDNA librarymixture and incubated at 42° C. for 26 hours. Hybrids between thebiotinylated oligo and the circular cDNA were isolated by diluting thehybridization mixture to 220 microliters in a solution containing 1 MNaCl, 10 mM Tris-HCl pH 7.5, 1 mM EDTA, pH 8.0 and adding 125microliters of streptavidin magnetic beads. This solution was incubatedat 42° C. for 60 mins, mixing every 5 mins to resuspend the beads. Thebeads were separated from the solution with a magnet and the beadswashed three times in 200 microliters of 0.1×SSPE, 0.1% SDS at 45° C.

The single stranded cDNAs were released from the biotinlyatedprobe/streptavidin magnetic bead complex by adding 50 microliters of 0.1N NaOH and incubating at room temperature for 10 mins. Six microlitersof 3 M Sodium Acetate was added along with 15 micrograms of glycogen andthe solution ethanol precipitated with 120 microliters of 100% ethanol.The DNA was resuspend in 12 microliters of TE (10 mM Tris-HCl, pH 8.0),1 mM EDTA, pH 8.0). The single stranded cDNA was converted into doublestrands in a thermal cycler by mixing 5 microliters of the captured DNAwith 1.5 microliters 10 micromolar standard SP6 primer (homologous to asequence on the cDNA cloning vector) and 1.5 microliters of 10×PCRbuffer. The mixture was heated to 95° C. for 20 seconds, then rampeddown to 59° C. At this time 15 microliters of a repair mix, that waspreheated to 70° C. (Repair mix contains 4 microliters of 5 mM dNTPs(1.25 mM each), 1.5 microliters of 10×PCR buffer, 9.25 microliters ofwater, and 0.25 microliters of Taq polymerase). The solution was rampedback to 73° C. and incubated for 23 mins. The repaired DNA was ethanolprecipitated and resuspended in 10 microliters of TE. Two microliterswere electroporated in E. coli DH12S cells and resulting colonies werescreened by PCR, using the following primer pair number:

Name Sequence Phos2-2s GAGAAAGCAGTCTTCCAGTTCTAC (SEQ ID NO: 156)Phos2-2a ATGGGAGCTAGAGGGTTTAATACT (SEQ ID NO: 157)

The sequence of the BMY_HPP2 polynucleotide was sequenced and isprovided in FIG. 21 (SEQ ID NO:151).

In the case of BMY_HPP5, BMY_HPP5 was cloned using the sequence ofIncyte clone 4155374 to design the following PCR oligos:

Oligo number Name Sequence 686 4155374-C3.s 5′-GGCCAAAGAGCAAACTCAAG-3(SEQ ID NO:69) 687 4155374-C3.Ba 5′-bGCATAGCTTGTTGGTCCCAT-3 (SEQ IDNO:70)

A biotinylated nucleotide was included on the 5′ end of oligo 687. Usingthe PCR primer pair, a 414 bp biotinylated fragment was amplified usingthe Incyte clone as the template. The fragment was gel purified byagarose electrophoresis and stored at 4° C. The same PCR primer pair wasused to screen cDNA libraries for the presence of homologous sequences.Positive PCR results were obtained in our HPLC-size fractionated brainand testis libraries. One microliter (one hundred and fifty nanograms)of the gel-purified biotinylated PCR fragment was added to sixmicroliters (six micrograms) of a single-stranded covalently closedcircular testis cDNA library and seven microliters of 100% formamide ina 0.5 ml PCR tube. The mixture was heated in a thermal cycler to 95° C.for 2 mins. Fourteen microliters of 2× hybridization buffer (50%formamide, 1.5 M NaCl, 0.04 M NaPO₄, pH 7.2, 5 mM EDTA, 0.2% SDS) wasadded to the heated probe/cDNA library mixture and incubated at 42° C.for 26 hours. Hybrids between the biotinylated oligo and the circularcDNA were isolated by diluting the hybridization mixture to 220microliters in a solution containing 1 M NaCl, 10 mM Tris-HCl pH 7.5, 1mM EDTA, pH 8.0 and adding 125 microliters of streptavidin magneticbeads. This solution was incubated at 42° C. for 60 mins, mixing every 5mins to resuspend the beads. The beads were separated from the solutionwith a magnet and the beads washed three times in 200 microliters of0.1×SSPE, 0.1% SDS at 45° C.

The single stranded cDNAs were released from the biotinlyatedprobe/streptavidin magnetic bead complex by adding 50 microliters of 0.1N NaOH and incubating at room temperature for 10 mins. Six microlitersof 3 M Sodium Acetate was added along with 15 micrograms of glycogen andthe solution ethanol precipitated with 120 microliters of 100% ethanol.The DNA was resuspend in 12 microliters of TE (10 mM Tris-HCl, pH 8.0),1 mM EDTA, pH 8.0). The single stranded cDNA was converted into doublestrands in a thermal cycler by mixing 5 microliters of the captured DNAwith 1.5 microliters 10 micromolar standard SP6 primer (homologous to asequence on the cDNA cloning vector) and 1.5 microliters of 10×PCRbuffer. The mixture was heated to 95° C. for 20 seconds, then rampeddown to 59° C. At this time 15 microliters of a repair mix, that waspreheated to 70° C. (Repair mix contains 4 microliters of 5 mM dNTPs(1.25 mM each), 1.5 microliters of 10×PCR buffer, 9.25 microliters ofwater, and 0.25 microliters of Taq polymerase). The solution was rampedback to 73° C. and incubated for 23 mins. The repaired DNA was ethanolprecipitated and resuspended in 10 microliters of TE. Two microliterswere electroporated in E. coli DH12S cells and resulting colonies werescreened by PCR, using the primer pair number 686/687. The sequence ofthe BMY_HPP5 polynucleotide was sequenced and is provided in FIGS. 5A-E(SEQ ID NO:41).

Example 3 Expression Profiling of the Novel Human BMY_HPP PhosphatasePolypeptides of the Present Invention

PCR primers designed from the predicted or observed cDNA sequences(described elsewhere herein) will be used in a real-time PCR assay todetermine relative steady state mRNA expression levels of BMY_HPP1,BMY_HPP2, BMY_HPP3, and BMY_HPP4 across a panel of human tissuesaccording to the following protocol.

First strand cDNA may be synthesized from commercially available mRNA(Clontech) and subjected to real time quantitative PCR using a PE 5700instrument (Applied Biosystems, Foster City, Calif.) using themanufacturers recommended protocol. This instrument detects the amountof DNA amplified during each cycle by the fluorescent output of SYBRgreen, a DNA binding dye specific for double strands. The specificity ofthe primer pair for its target may be verified by performing a thermaldenaturation profile at the end of the run which provides an indicationof the number of different DNA sequences present by determining meltingTm. Only primer pairs giving a single PCR product are considered.Contributions of contaminating genomic DNA to the assessment of tissueabundance may be controlled by performing the PCR with first strand madewith and without reverse transcriptase. Only samples where thecontribution of material amplified in the no reverse transcriptasecontrols was negligible are considered.

Small variations in the amount of cDNA used in each tube can bedetermined by performing a parallel experiment using a primer pair forthe cyclophilin gene, which is expressed in equal amounts in alltissues. The data is then used to normalize the data obtained with eachprimer pair. The PCR data was converted into a relative assessment ofthe difference in transcript abundance amongst the tissues tested.

Representative primers for expression profiling analysis for each geneare provided in Table VI herein as ‘EP Sense’ and ‘EP Anti-sensePrimer’, though may also include one or more of the following: ‘LeftCloning Primer’, ‘Right Cloning Primer’, ‘Internal RevComp CloningPrimer’, and/or ‘Internal Cloning Primer’. Other primers could besubsitituted for any of the above as would be appreciated by one skilledin the art.

In the case of BMY_HPP1, the following PCR primer pair was used tomeasure the steady state levels of BMY_HPP1 mRNA by quantitative PCR:

Sense: 5′- TACAATTTCGGATGGAAGGATTAT -3′ (SEQ ID NO:154) Antisense: 5′-GCATGACAATGGATAGCTACTTT -3′ (SEQ ID NO:155)

Briefly, first strand cDNA was made from commercially available mRNA.The relative amount of cDNA used in each assay was determined byperforming a parallel experiment using a primer pair for a geneexpressed in equal amounts in all tissues, cyclophilin. The cyclophilinprimer pair detected small variations in the amount of cDNA in eachsample and these data were used for normalization of the data obtainedwith the primer pair for the novel BMY_HPP1. The PCR data was convertedinto a relative assessment of the difference in transcript abundanceamongst the tissues tested and the data is presented in FIG. 22.Transcripts corresponding to BMY_HPP1 were expressed highly in testis;to a significant extent, in the spinal cord, and to a lesser extent, inpancreas, brain, pituitary, heart, and lung.

In the case of BMY_HPP2, the following PCR primer pair was used tomeasure the steady state levels of BMY_HPP2 mRNA by quantitative PCR:

Sense: 5′- GAGAAAGCAGTCTTCCAGTTCTAC -3′ (SEQ ID NO:156) Antisense: 5′-ATGGGAGCTAGAGGGTTTAATACT -3′ (SEQ ID NO:157)

Briefly, first strand cDNA was made from commercially available mRNA.The relative amount of cDNA used in each assay was determined byperforming a parallel experiment using a primer pair for a geneexpressed in equal amounts in all tissues, cyclophilin. The cyclophilinprimer pair detected small variations in the amount of cDNA in eachsample and these data were used for normalization of the data obtainedwith the primer pair for the novel BMY_HPP2. The PCR data was convertedinto a relative assessment of the difference in transcript abundanceamongst the tissues tested and the data is presented in FIG. 23.Transcripts corresponding to BMY_HPP2 were expressed highly in liver andkidney; to a significant extent, in the spleen, and to a lesser extent,in lung, testis, heart, intestine, pancreas, lymph node, spinal cord,and prostate.

In the case of BMY_HPP5, the following PCR primer pair was used tomeasure the steady state levels of BMY_HPP5 mRNA by quantitative PCR:

Sense: 5′- ATGGGACCAACAAGCTATGC -3′ (SEQ ID NO:67) Antisense: 5′-TTATCAGGACTGGTTTCGGG -3′ (SEQ ID NO:68)

Briefly, first strand cDNA was made from commercially available mRNA.The relative amount of cDNA used in each assay was determined byperforming a parallel experiment using a primer pair for a geneexpressed in equal amounts in all tissues, cyclophilin. The cyclophilinprimer pair detected small variations in the amount of cDNA in eachsample and these data were used for normalization of the data obtainedwith the primer pair for the novel BMY_HPP5. The PCR data was convertedinto a relative assessment of the difference in transcript abundanceamongst the tissues tested and the data is presented in FIG. 11.Transcripts corresponding to BMY_HPP5 were expressed highly in thetestis, spinal cord, an to a lesser extent in bone marrow, brain,thymus, and liver.

Example 4 Method of Assaying the Phosphatase Activity of the BMY_HPPPolypeptides of the Present Invention

The Phosphatase Activity of the BMY_HPP Polypeptides of the presentinvention may be assessed through the application of various biochemicalassays known in the art and described herein.

Hydrolysis of Para-Nitrophenyl Phosphate

The phosphatase activity for BMY_HPP proteins may be measured byassaying the ability of the proteins to hydrolize para-nitrophenylphosphate, a compound known to be a substrate for phosphatases, asdescribed in Krejsa, C. et al., J. Biol. Chem. Vol. 272, p. 1541-11549,1997 (which is hereby incorporated in its entirety herein). The proteinsare incubated in 3 mg/ml para-nitrophenyl phosphate in a solutioncontaining 60 mM MES, pH 6.0, 5% glycerol, 5 mM dithiothreitol, and 0.1%Triton X-100 for 15 min, or such other time as may be desired. The pH ofthe reaction may be varied to provide an optimal pH for each individualBMY_HPP protein by those with ordinary skill in the art of enzymeassays. The phosphatase reaction is stopped by the addition of 3 N NaOHto give a final NaOH concentration of 0.7 M. The product of the reactionis measured by reading the absorbance of the solution at 405 nm.

Two Dimensional Gel Electrophoresis

The BMY_HPP polynucleotides of the present invention may be subclonedinto appropriate vectors for expression in host cells. Representativevectors are known in the art and described herein. 2-D gelelectrophoresis (IEF followed by SDS-PAGE) will be used to assayBMY_HPP-dependent dephosphorylation of host cell proteins. Theseproteins can be recovered from the gel and identified by massspectrometric or other protein sequencing techniques known in the art.

Briefly, Methods for 2-dimensional gel analysis and labeling cells withproteins are well known in the art. Cells would be labeled with 32Porthophosphate, cellular proteins would be resolved on 2D gels and theirpositions determined by autoradiography. Proteins of interest would beidentified by excising the spots and analyzing their sequence by massspectroscopy. The following paper and the references therein describethe methods of labeling cells, analyzing the proteins on 2D gels andmass spec identification: Gerner, C. et al., J. Biol. Chem., Vol. 275,p. 39018-39026, 2000. Substrates affected by the phosphatase would beidentified by comparing wild type cells to cells where expression of thephosphatase is inhibited by deletion, anti-sense, or other means.Proteins whose phosphorylation increased would be either directsubstrates or indirectly regulated by the phosphatase. Conversely, incells where the active phosphatase was overexpressed, proteins whosephosphorylation decreased would either be direct substrates orindirectly regulated by the phosphatase.

Example 5 Method of Identifying the Substrate of the BMY_HPP PhosphatasePolypeptides of the Present Invention

Substrate Identification

Protein substrates for BMY_HPP polypeptides of the present invention maybe identified by recovery of proteins dephosphorylated in the 2-D gelelectrophoesis assay described above. Phosphopeptide substrates may alsobe identified as proteins whose phosphorylation increases when theactivity or expression of a BMY_HPP protein is decreased (for example,by an antibody, antisense or double-stranded inhibitory RNA or by asmall moloecule inhibitor of BMY_HPP activity). In either case, massspectrometry can be used to identify the recovered proteins.

Phosphopeptide substrates for BMY-HPP polypeptides may also beidentified by incubation of a phosphopeptide library with acatalytically inactive version of the protein, recovery of the complex,and peptide sequencing by standard methods such as Edman degradation ormass spectrometry.

Phosphopeptide substrates can also be identified by expressing asubstrate trapping mutant phosphatase (one that is catalyticallyinactive due to active site mutation) and isolating the proteins thatbind preferentially to the substrate trapping phosphatase relative tothe wild type phosphatase.

Example 6 Method of Identifying RET31 of the Present Invention

In an effort to identify gene products involved in regulatory events,the RNA expressed in TNF-α-stimulated human lung microvascularendothelial cells was analyzed. Resting cells were stimulated for 1 hwith TNF-α, and the RNA was isolated from the cells. Complementary DNA(cDNA) was created from the isolated RNA using methods known in the art.The cDNA that were upregulated in response to TNFα were identified usingsubtractive hybridization. A clone corresponding to a portion of theRET31 polynucleotide was identified and used to identify the full-length(SEQ ID NO:115). Additional methods are provided below.

HMVEC Cell Culture

Primary cultures of human lung microvascular endothelial cells (HMVEC),from a single donor, were obtained from Clonetics (Walkersville, Md.).The cells were grown in the endothelial cell growth medium-2 kit(CC-3202) with 5% Fetal Bovine Serum (Hyclone). Initially, the cellswere seeded into a T-25 flask and, after reaching approximately 90%confluence, they were trypsinized and transferred into T-225 flasks at1.2×10⁶/flask in 80 mls of medium. For normal growth conditions, themedium was changed each 48 h. When the cells reached approximately 90%confluence, they were passaged again and seeded into T-225 flasks at1.8×10⁶/ml in 80 mls of medium.

HMVEC Cell Treatment for RNA Isolation

Subconfluent (90% confluent) T-225 flasks of HMVEC were adjusted to 40ml of medium per flask by removing excess medium. HMVEC were either leftuntreated (time 0) or treated with 10 ng/ml TNF-α for 1, 6 or 24 h. Themedium was not changed at the time of TNF-α addition.

RNA Isolation

At the designated time points, The flasks of HMVEC were trypsinizedbriefly to remove cells from the flasks and trypsinization wasterminated by the addition of fetal calf serum. The cells were removedfrom the flasks and the flasks rinsed with PBS. The cells were pelleted,rinsed once in PBS and re-pelleted. The supernatant was removed and thecell pellet used for RNA isolation. Poly A+ RNA was isolated directlyusing Fast Track 2.0™ (Invitrogen, Carlsbad, Calif.).

Construction of the Subtraction Library

The PCR-select cDNA subtraction kit™ (Clontech, Palo Alto, Calif.) wasused to generate a subtraction library from untreated HMVEC poly A+ RNA(tester) and 1 h TNF-α-treated HMVEC poly A+ RNA (driver), according tothe manufacturer's protocols. Ten secondary PCR reactions were combinedand run on a 2% agarose gel. Fragments ranging from approximately 0.3kb-10 kb were gel purified using the QIAgen gel extraction kit (QIAgenInc., Valencia, Calif.) and inserted into the TA cloning vector, pCR2.1(Invitrogen). TOP10F′ competent E. coli (Invitrogen) were transformedand plated on LB plates containing 50 micrograms/ml ampicillin. Cloneswere isolated and grown in LB broth containing similar concentrations ofampicillin. Plasmids were sequenced using methods known in the art ordescribed elsewhere herein.

As referenced above, the methods utilized for constructing thesubtraction library followed the PCR-Select cDNA Subtraction Kit(Clonetech; Protocol # PT1117-1; Version # PR85431) which is herebyincorporated herein by reference in its entirety. Additional referencesto this method may be found in Diatchenko, L., et al., PNAS 93:6025-6030(1996), which is hereby incorporated herein by reference in itsentirety.

Example 7 Method of Cloning RET31 of the Present Invention

A clone containing the predicted coding sequence of RET31 was isolatedfrom human microvascular endothelial cells (HMVECs) treated with tumornecrosis factor alpha (TNFα) for 6 hours using reversetranscription/polymerase chain reaction (RT/PCR). RNA was purified fromthe TNFa stimulated HMVEC cells according to methods known in the art. Aprimer set (each at 400 nM final concentration) was used to amplify a 3kb sequence using the following primers and conditions:

primer JNF388: CACACCACCATTACATCATCGTGGC (SEQ ID NO:145) primer JNF525:TGCTGCTCTGCTACCAACCC (SEQ ID NO:146)with 200 μM dNTP's, 1× Advantage 2 polymerase, and 2.0 μl DNA in 25.0 μlreaction. The experiment was cycled 35 times through the followingsequence: 94° C. for 30 sec, 68° C. for 30 sec. then 72° C. for 3.5 min.At the completion of the reaction, 6.0 μl of loading dye was added andthe entire reaction was separated by gel electrophoresis in a 1.2%agarose gel containing ethidium bromide. An ˜3 kb size band was excisedfrom the gel and purified using the QIAgen extraction kit (QIAgen,Valencia, Calif.). This fragment was ligated into the pTAdv cloningvector (Clontech, Palo Alto, Calif.) and sequenced using standardmethods. The RET31 clone (SEQ ID NO:108; FIGS. 13A-F) contains about a 3kb sequence corresponding to nucleotides 472 to 3513 of the predictedRET31 coding sequence (SEQ ID NO:147). The predicted RET31 codingsequence (SEQ ID NO:147) was derived from Incyte gene cluster 1026659.7.

A nucleic acid sequence corresponding to the nucleic acid sequenceencoding the RET31 polypeptide was first identified in a subtractionlibrary from TNF-α stimulated human lung microvascular endiothelialcells (HMVEC). This subtraction clone sequence encoded a 408 bp partialcDNA sequence, as shown:

RET31 Subtraction Clone

(SEQ ID NO:115) ACAATGGAGTGGCTGAGCCTTTGAGCACACCACCATTACATCATCGTGGCAAATTAAAGAAGGAGGTGGGAAAAGAGGACTTATTGTTGTCATGGCCCATGAGATGATTGGAACTCAAATTGTTACTGAGAGGTTGGTGGCTCTGCTGGAAAGTGGAACGGAAAAAGTGCTGCTAATTGATAGCCGGCCATTTGTGGAATACAATACATCCCACATTTTGGAAGCCATTAATATCAACTGCTCCAAGCTTATGAAGCGAAGGTTGCAACAGGACAAAGTGTTAATTACAGAGCTCATCCAGCATTCAGCGAAACATAAGGTTGACATTGATTGCAGTCAGAAGGTTGTAGTTTACGATCAAAGCTCCCAAGATGTTGCCTCTCTCTCTTCAGACTGTTTT CTCACTGT

Example 8 Method of Determining the mRNA Expression Profile of RET31Using Northern Hybidization

Multiple tissue northern blots (MTN) were purchased from ClontechLaboratories (Palo Alto, Calif.) and hybridized with P³²-labeled RET31.Briefly, a 408 bp RET31 fragment (RET31/RsaI) was isolated fromsubtraction clone 1hrTNF031 (SEQ ID NO:115) using RsaI restrictionendonuclease, run on a 2.0% agarose gel, and purified using the QIAgenGel Extraction Kit (QIAgen, Valencia, Calif.). Approximately 30 ng ofRET31/RsaI was radiolabeled (6000 Ci/mmol P³²-dCTP) using the RandomPrimed DNA Labeling Kit (Roche, Indianapolis, Ind.). Unincorporatednucleotides were removed using NucTrap Probe Purification Columns(Stratgene, La Jolla, Calif.). Radiolabeled RET31/RsaI probe was addedat a specific activity of 1.5×10⁶ cpm/ml of ExpressHyb hybridizationsolution (Clontech) and incubated overnight at 65° C. Blots were washedto 2.0×SSC/0.05% SDS at 50° C. and exposed to film for 24 and 48 h. TheMTN's used were human MTN (#7760-1), human MTN II (#7759-1), human MTNIII (#7767-1), and human cancer cell line MTN (#7757-1).

The results show the RET31 polypeptide was expressed predominately inadrenal gland, testis, and skeletal muscle; to a significant extent, inthe liver, prostate ovary, and to a lesser extent, in placenta,pancreas, thymus, small intestine, thyroid, heart, kidney and liver (seeFIG. 15).

Example 9 Method of Assessing the Affect of TNF-Alpha on RET31 mRNAExpression

In an effort to confirm the the TNF-alpha dependent regulation of RET31expression, HMVEC cells were treated with TNF-alpha over several timeperiods and the mRNA subsequently harvested and probed by northernhybridization. Briefly, untreated HMVEC, 1 h TNF-α stimulated HMVEC, 6 hTNF-α stimulated HMVEC, 24 h TNF-α stimulated HMVEC poly A+ RNA (2 μgeach) were run on a 1.2% agarose gel containing 3.0% formaldehyde andtransferred to Hybond N+ nucleic acid transfer membrane (Amersham,Piscataway, N.J.) using standard blotting techniques (see Maniatis etal. referenced herein). Membranes were auto cross-linked usingStratalinker (Stratagene) and prehybridized in ExpressHyb hybridizationsolution for 1 h and probed in parallel with the multiple tissuenorthern blots.

After hybridization, membranes were washed by continuous shaking for 30minutes with low stringency solution (2×SSC/0.05% SDS) at roomtemperature with 2 changes of solution. Membranes were then washed for30 minutes with high stringency solution (0.1×SSC/0.1% SDS) at 50° C.with 1 change of solution. The membranes were exposed with intensifyingscreens to X-ray film at −70° C. for 10 days.

The endothelial cell blot was reprobed for E-selectin and GAPDH.

The results confirmed RET31 is up-regulated by TNF-α, reaching a peak ofexpression at 6 h by northern blot analysis (see FIG. 18).

Example 6 Method of Assessing the Physiological Function of the humanPhosphatase Polypeptide at the Cellular Level

The physiological function of the human phosphatase polypeptide may beassessed by expressing the sequences encoding human phosphatase atphysiologically elevated levels in mammalian cell culture systems. cDNAis subcloned into a mammalian expression vector containing a strongpromoter that drives high levels of cDNA expression (examples areprovided elsewhere herein). Vectors of choice include pCMV SPORT (LifeTechnologies) and pCR3.1 (Invitrogen, Carlsbad Calif.), both of whichcontain the cytomegalovirus promoter. 5-10, ug of recombinant vector aretransiently transfected into a human cell line, preferably ofendothelial or hematopoietic origin, using either liposome formulationsor electroporation. 1-2 ug of an additional plasmid containing sequencesencoding a marker protein are cotransfected. Expression of a markerprotein provides a means to distinguish transfected cells fromnontransfected cells and is a reliable predictor of cDNA expression fromthe recombinant vector. Marker proteins of choice include, e.g., GreenFluorescent Protein (GFP; Clontech), CD64, or a CD64-GFP fusion protein.Flow cytometry (FCM), an automated, laser optics-based technique, isused to identify transfected cells expressing GFP or CD64-GFP and toevaluate the apoptotic state of the cells and other cellular properties.FCM detects and quantifies the uptake of fluorescent molecules thatdiagnose events preceding or coincident with cell death. These eventsinclude changes in nuclear DNA content as measured by staining of DNAwith propidium iodide; changes in cell size and granularity as measuredby forward light scatter and 90 degree side light scatter;down-regulation of DNA synthesis as measured by decrease inbromodeoxyuridine uptake; alterations in expression of cell surface andintracellular proteins as measured by reactivity with specificantibodies; and alterations in plasma membrane composition as measuredby the binding of fluorescein-conjugated Annexin V protein to the cellsurface. Methods in flow cytometry are discussed in Ormerod, M. G.(1994) Flow Cvtometrv, Oxford, New York N.Y.

The influence of human phosphatase polypeptides on gene expression canbe assessed using highly purified populations of cells transfected withsequences encoding human phosphatase and either CD64 or CD64-GFP. CD64and CD64-GFP are expressed on the surface of transfected cells and bindto conserved regions of human immunoglobulin G (IgG). Transfected cellsare efficiently separated from nontransfected cells using magnetic beadscoated with either human IgG or antibody against CD64 (DYNAL, LakeSuccess N.Y.). mRNA can be purified from the cells using methods wellknown by those of skill in the art. Expression of mRNA encoding humanphosphatase polypeptides and other genes of interest can be analyzed bynorthern analysis or microarray techniques.

Example 7 Method of Screening for Compounds that Interact with the HumanPhosphatase Polypeptide

The following assays are designed to identify compounds that bind to thehuman phosphatase polypeptide, bind to other cellular proteins thatinteract with the human phosphatase polypeptide, and to compounds thatinterfere with the interaction of the human phosphatase polypeptide withother cellular proteins.

Such compounds can include, but are not limited to, other cellularproteins. Specifically, such compounds can include, but are not limitedto, peptides, such as, for example, soluble peptides, including, but notlimited to Ig-tailed fusion peptides, comprising extracellular portionsof human phosphatase polypeptide transmembrane receptors, and members ofrandom peptide libraries (see, e.g., Lam, K. S. et al., 1991, Nature354:82-84; Houghton, R. et al., 1991, Nature 354:84-86), made of D-and/or L-configuration amino acids, phosphopeptides (including, but notlimited to, members of random or partially degenerate phosphopeptidelibraries; see, e.g., Songyang, Z., et al., 1993, Cell 72:767-778),antibodies (including, but not limited to, polyclonal, monoclonal,humanized, anti-idiotypic, chimeric or single chain antibodies, and FAb,F(ab′).sub.2 and FAb expression libary fragments, and epitope-bindingfragments thereof), and small organic or inorganic molecules.

Compounds identified via assays such as those described herein can beuseful, for example, in elaborating the biological function of the humanphosphatase polypeptide, and for ameliorating symptoms of tumorprogression, for example. In instances, for example, whereby a tumorprogression state or disorder results from a lower overall level ofhuman phosphatase expression, human phosphatase polypeptide, and/orhuman phosphatase polypeptide activity in a cell involved in the tumorprogression state or disorder, compounds that interact with the humanphosphatase polypeptide can include ones which accentuate or amplify theactivity of the bound human phosphatase polypeptide. Such compoundswould bring about an effective increase in the level of humanphosphatase polypeptide activity, thus ameliorating symptoms of thetumor progression disorder or state. In instances whereby mutationswithin the human phosphatase polypeptide cause aberrant humanphosphatase polypeptides to be made which have a deleterious effect thatleads to tumor progression, compounds that bind human phosphatasepolypeptide can be identified that inhibit the activity of the boundhuman phosphatase polypeptide. Assays for testing the effectiveness ofsuch compounds are known in the art and discussed, elsewhere herein.

Example 8 Method of Screening, In Vitro, Compounds that Bind to theHuman Phosphatase Polypeptide

In vitro systems can be designed to identify compounds capable ofbinding the human phosphatase polypeptide of the invention. Compoundsidentified can be useful, for example, in modulating the activity ofwild type and/or mutant human phosphatase polypeptide, preferably mutanthuman phosphatase polypeptide, can be useful in elaborating thebiological function of the human phosphatase polypeptide, can beutilized in screens for identifying compounds that disrupt normal humanphosphatase polypeptide interactions, or can in themselves disrupt suchinteractions.

The principle of the assays used to identify compounds that bind to thehuman phosphatase polypeptide involves preparing a reaction mixture ofthe human phosphatase polypeptide and the test compound under conditionsand for a time sufficient to allow the two components to interact andbind, thus forming a complex which can be removed and/or detected in thereaction mixture. These assays can be conducted in a variety of ways.For example, one method to conduct such an assay would involve anchoringhuman phosphatase polypeptide or the test substance onto a solid phaseand detecting human phosphatase polypeptide/test compound complexesanchored on the solid phase at the end of the reaction. In oneembodiment of such a method, the human phosphatase polypeptide can beanchored onto a solid surface, and the test compound, which is notanchored, can be labeled, either directly or indirectly.

In practice, microtitre plates can conveniently be utilized as the solidphase. The anchored component can be immobilized by non-covalent orcovalent attachments. Non-covalent attachment can be accomplished bysimply coating the solid surface with a solution of the protein anddrying. Alternatively, an immobilized antibody, preferably a monoclonalantibody, specific for the protein to be immobilized can be used toanchor the protein to the solid surface. The surfaces can be prepared inadvance and stored.

In order to conduct the assay, the nonimmobilized component is added tothe coated surface containing the anchored component. After the reactionis complete, unreacted components are removed (e.g., by washing) underconditions such that any complexes formed will remain immobilized on thesolid surface. The detection of complexes anchored on the solid surfacecan be accomplished in a number of ways. Where the previouslyimmobilized component is pre-labeled, the detection of label immobilizedon the surface indicates that complexes were formed. Where thepreviously nonimmobilized component is not pre-labeled, an indirectlabel can be used to detect complexes anchored on the surface; e.g.,using a labeled antibody specific for the immobilized component (theantibody, in turn, can be directly labeled or indirectly labeled with alabeled anti-Ig antibody).

Alternatively, a reaction can be conducted in a liquid phase, thereaction products separated from unreacted components, and complexesdetected; e.g., using an immobilized antibody specific for humanphosphatase polypeptide or the test compound to anchor any complexesformed in solution, and a labeled antibody specific for the othercomponent of the possible complex to detect anchored complexes.

Example 9 Method of Identifying Compounds that Interfere with HumanPhosphatase Polypeptide/Cellular Product Interaction

The human phosphatase polypeptide of the invention can, in vivo,interact with one or more cellular or extracellular macromolecules, suchas proteins. Such macromolecules include, but are not limited to,polypeptides, particularly ligands, and those products identified viascreening methods described, elsewhere herein. For the purposes of thisdiscussion, such cellular and extracellular macromolecules are referredto herein as “binding partner(s)”. For the purpose of the presentinvention, “binding partner” may also encompass polypeptides, smallmolecule compounds, polysaccarides, lipids, and any other molecule ormolecule type referenced herein. Compounds that disrupt suchinteractions can be useful in regulating the activity of the humanphosphatase polypeptide, especially mutant human phosphatasepolypeptide. Such compounds can include, but are not limited tomolecules such as antibodies, peptides, and the like described inelsewhere herein.

The basic principle of the assay systems used to identify compounds thatinterfere with the interaction between the human phosphatase polypeptideand its cellular or extracellular binding partner or partners involvespreparing a reaction mixture containing the human phosphatasepolypeptide, and the binding partner under conditions and for a timesufficient to allow the two products to interact and bind, thus forminga complex. In order to test a compound for inhibitory activity, thereaction mixture is prepared in the presence and absence of the testcompound. The test compound can be initially included in the reactionmixture, or can be added at a time subsequent to the addition of humanphosphatase polypeptide and its cellular or extracellular bindingpartner. Control reaction mixtures are incubated without the testcompound or with a placebo. The formation of any complexes between thehuman phosphatase polypeptide and the cellular or extracellular bindingpartner is then detected. The formation of a complex in the controlreaction, but not in the reaction mixture containing the test compound,indicates that the compound interferes with the interaction of the humanphosphatase polypeptide and the interactive binding partner.Additionally, complex formation within reaction mixtures containing thetest compound and normal human phosphatase polypeptide can also becompared to complex formation within reaction mixtures containing thetest compound and mutant human phosphatase polypeptide. This comparisoncan be important in those cases wherein it is desirable to identifycompounds that disrupt interactions of mutant but not normal humanphosphatase polypeptide.

The assay for compounds that interfere with the interaction of the humanphosphatase polypeptide and binding partners can be conducted in aheterogeneous or homogeneous format. Heterogeneous assays involveanchoring either the human phosphatase polypeptide or the bindingpartner onto a solid phase and detecting complexes anchored on the solidphase at the end of the reaction. In homogeneous assays, the entirereaction is carried out in a liquid phase. In either approach, the orderof addition of reactants can be varied to obtain different informationabout the compounds being tested. For example, test compounds thatinterfere with the interaction between the human phosphatase polypeptideand the binding partners, e.g., by competition, can be identified byconducting the reaction in the presence of the test substance; i.e., byadding the test substance to the reaction mixture prior to orsimultaneously with the human phosphatase polypeptide and interactivecellular or extracellular binding partner. Alternatively, test compoundsthat disrupt preformed complexes, e.g. compounds with higher bindingconstants that displace one of the components from the complex, can betested by adding the test compound to the reaction mixture aftercomplexes have been formed. The various formats are described brieflybelow.

In a heterogeneous assay system, either the human phosphatasepolypeptide or the interactive cellular or extracellular bindingpartner, is anchored onto a solid surface, while the non-anchoredspecies is labeled, either directly or indirectly. In practice,microtitre plates are conveniently utilized. The anchored species can beimmobilized by non-covalent or covalent attachments. Non-covalentattachment can be accomplished simply by coating the solid surface witha solution of the human phosphatase polypeptide or binding partner anddrying. Alternatively, an immobilized antibody specific for the speciesto be anchored can be used to anchor the species to the solid surface.The surfaces can be prepared in advance and stored.

In order to conduct the assay, the partner of the immobilized species isexposed to the coated surface with or without the test compound. Afterthe reaction is complete, unreacted components are removed (e.g., bywashing) and any complexes formed will remain immobilized on the solidsurface. The detection of complexes anchored on the solid surface can beaccomplished in a number of ways. Where the non-immobilized species ispre-labeled, the detection of label immobilized on the surface indicatesthat complexes were formed. Where the non-immobilized species is notpre-labeled, an indirect label can be used to detect complexes anchoredon the surface; e.g., using a labeled antibody specific for theinitially non-immobilized species (the antibody, in turn, can bedirectly labeled or indirectly labeled with a labeled anti-Ig antibody).Depending upon the order of addition of reaction components, testcompounds which inhibit complex formation or which disrupt preformedcomplexes can be detected.

Alternatively, the reaction can be conducted in a liquid phase in thepresence or absence of the test compound, the reaction productsseparated from unreacted components, and complexes detected; e.g., usingan immobilized antibody specific for one of the binding components toanchor any complexes formed in solution, and a labeled antibody specificfor the other partner to detect anchored complexes. Again, dependingupon the order of addition of reactants to the liquid phase, testcompounds which inhibit complex or which disrupt preformed complexes canbe identified.

In an alternate embodiment of the invention, a homogeneous assay can beused. In this approach, a preformed complex of the human phosphatasepolypeptide and the interactive cellular or extracellular bindingpartner product is prepared in which either the human phosphatasepolypeptide or their binding partners are labeled, but the signalgenerated by the label is quenched due to complex formation (see, e.g.,U.S. Pat. No. 4,109,496 by Rubenstein which utilizes this approach forimmunoassays). The addition of a test substance that competes with anddisplaces one of the species from the preformed complex will result inthe generation of a signal above background. In this way, testsubstances which disrupt human phosphatase polypeptide-cellular orextracellular binding partner interaction can be identified.

In a particular embodiment, the human phosphatase polypeptide can beprepared for immobilization using recombinant DNA techniques known inthe art. For example, the human phosphatase polypeptide coding regioncan be fused to a glutathione-5-transferase (GST) gene using a fusionvector such as pGEX-5X-1, in such a manner that its binding activity ismaintained in the resulting fusion product. The interactive cellular orextracellular product can be purified and used to raise a monoclonalantibody, using methods routinely practiced in the art and describedabove. This antibody can be labeled with the radioactive isotope.sup.125 I, for example, by methods routinely practiced in the art. In aheterogeneous assay, e.g., the GST-human phosphatase polypeptide fusionproduct can be anchored to glutathione-agarose beads. The interactivecellular or extracellular binding partner product can then be added inthe presence or absence of the test compound in a manner that allowsinteraction and binding to occur. At the end of the reaction period,unbound material can be washed away, and the labeled monoclonal antibodycan be added to the system and allowed to bind to the complexedcomponents. The interaction between the human phosphatase polypeptideand the interactive cellular or extracellular binding partner can bedetected by measuring the amount of radioactivity that remainsassociated with the glutathione-agarose beads. A successful inhibitionof the interaction by the test compound will result in a decrease inmeasured radioactivity.

Alternatively, the GST-human phosphatase polypeptide fusion product andthe interactive cellular or extracellular binding partner product can bemixed together in liquid in the absence of the solid glutathione-agarosebeads. The test compound can be added either during or after the bindingpartners are allowed to interact. This mixture can then be added to theglutathione-agarose beads and unbound material is washed away. Again theextent of inhibition of the binding partner interaction can be detectedby adding the labeled antibody and measuring the radioactivityassociated with the beads.

In another embodiment of the invention, these same techniques can beemployed using peptide fragments that correspond to the binding domainsof the human phosphatase polypeptide product and the interactivecellular or extracellular binding partner (in case where the bindingpartner is a product), in place of one or both of the full lengthproducts.

Any number of methods routinely practiced in the art can be used toidentify and isolate the protein's binding site. These methods include,but are not limited to, mutagenesis of one of the genes encoding one ofthe products and screening for disruption of binding in aco-immunoprecipitation assay. Compensating mutations in the geneencoding the second species in the complex can be selected. Sequenceanalysis of the genes encoding the respective products will reveal themutations that correspond to the region of the product involved ininteractive binding. Alternatively, one product can be anchored to asolid surface using methods described in this Section above, and allowedto interact with and bind to its labeled binding partner, which has beentreated with a proteolytic enzyme, such as trypsin. After washing, ashort, labeled peptide comprising the binding domain can remainassociated with the solid material, which can be isolated and identifiedby amino acid sequencing. Also, once the gene coding for the cellular orextracellular binding partner product is obtained, short gene segmentscan be engineered to express peptide fragments of the product, which canthen be tested for binding activity and purified or synthesized.

Example 10 Isolation of a Specific Clone from the Deposited Sample

The deposited material in the sample assigned the ATCC Deposit Numbercited in Table I for any given cDNA clone also may contain one or moreadditional plasmids, each comprising a cDNA clone different from thatgiven clone. Thus, deposits sharing the same ATCC Deposit Number containat least a plasmid for each cDNA clone identified in Table I. Typically,each ATCC deposit sample cited in Table I comprises a mixture ofapproximately equal amounts (by weight) of about 1-10 plasmid DNAs, eachcontaining a different cDNA clone and/or partial cDNA clone; but such adeposit sample may include plasmids for more or less than 2 cDNA clones.

Two approaches can be used to isolate a particular clone from thedeposited sample of plasmid DNA(s) cited for that clone in Table I.First, a plasmid is directly isolated by screening the clones using apolynucleotide probe corresponding to SEQ ID NO:X.

Particularly, a specific polynucleotide with 30-40 nucleotides issynthesized using an Applied Biosystems DNA synthesizer according to thesequence reported. The oligonucleotide is labeled, for instance, with32P-(-ATP using T4 polynucleotide kinase and purified according toroutine methods. (E.g., Maniatis et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Press, Cold Spring, N.Y. (1982).) The plasmidmixture is transformed into a suitable host, as indicated above (such asXL-1 Blue (Stratagene)) using techniques known to those of skill in theart, such as those provided by the vector supplier or in relatedpublications or patents cited above. The transformants are plated on1.5% agar plates (containing the appropriate selection agent, e.g.,ampicillin) to a density of about 150 transformants (colonies) perplate. These plates are screened using Nylon membranes according toroutine methods for bacterial colony screening (e.g., Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Edit., (1989), Cold SpringHarbor Laboratory Press, pages 1.93 to 1.104), or other techniques knownto those of skill in the art.

Alternatively, two primers of 17-20 nucleotides derived from both endsof the SEQ ID NO:X (i.e., within the region of SEQ ID NO:X bounded bythe 5′ NT and the 3′ NT of the clone defined in Table I) are synthesizedand used to amplify the desired cDNA using the deposited cDNA plasmid asa template. The polymerase chain reaction is carried out under routineconditions, for instance, in 25 ul of reaction mixture with 0.5 ug ofthe above cDNA template. A convenient reaction mixture is 1.5-5 mMMgCl2, 0.01% (w/v) gelatin, 20 uM each of dATP, dCTP, dGTP, dTTP, 25pmol of each primer and 0.25 Unit of Taq polymerase. Thirty five cyclesof PCR (denaturation at 94 degree C. for 1 min; annealing at 55 degreeC. for 1 min; elongation at 72 degree C. for 1 min) are performed with aPerkin-Elmer Cetus automated thermal cycler. The amplified product isanalyzed by agarose gel electrophoresis and the DNA band with expectedmolecular weight is excised and purified. The PCR product is verified tobe the selected sequence by subcloning and sequencing the DNA product.

Example 11 Tissue Distribution of Polypeptide

Tissue distribution of mRNA expression of polynucleotides of the presentinvention is determined using protocols for Northern blot analysis,described by, among others, Sambrook et al. For example, a cDNA probeproduced by the method described in Example 10 is labeled with p32 usingthe rediprime™ DNA labeling system (Amersham Life Science), according tomanufacturer's instructions. After labeling, the probe is purified usingCHROMA SPIN0-100 column (Clontech Laboratories, Inc.) according tomanufacturer's protocol number PT1200-1. The purified labeled probe isthen used to examine various tissues for mRNA expression.

Tissue Northern blots containing the bound mRNA of various tissues areexamined with the labeled probe using ExpressHyb™ hybridization solution(Clonetech according to manufacturers protocol number PT1190-1. Northernblots can be produced using various protocols well known in the art(e.g., Sambrook et al). Following hybridization and washing, the blotsare mounted and exposed to film at −70 C overnight, and the filmsdeveloped according to standard procedures.

Example 12 Chromosomal Mapping of the Polynucleotides

An oligonucleotide primer set is designed according to the sequence atthe 5′ end of SEQ ID NO:X. This primer preferably spans about 100nucleotides. This primer set is then used in a polymerase chain reactionunder the following set of conditions: 30 seconds, 95 degree C.; 1minute, 56 degree C.; 1 minute, 70 degree C. This cycle is repeated 32times followed by one 5 minute cycle at 70 degree C. Mammalian DNA,preferably human DNA, is used as template in addition to a somatic cellhybrid panel containing individual chromosomes or chromosome fragments(Bios, Inc). The reactions are analyzed on either 8% polyacrylamide gelsor 3.5% agarose gels. Chromosome mapping is determined by the presenceof an approximately 100 bp PCR fragment in the particular somatic cellhybrid.

Example 13 Bacterial Expression of a Polypeptide

A polynucleotide encoding a polypeptide of the present invention isamplified using PCR oligonucleotide primers corresponding to the 5′ and3′ ends of the DNA sequence, as outlined in Example 10, to synthesizeinsertion fragments. The primers used to amplify the cDNA insert shouldpreferably contain restriction sites, such as BamHI and XbaI, at the 5′end of the primers in order to clone the amplified product into theexpression vector. For example, BamHI and XbaI correspond to therestriction enzyme sites on the bacterial expression vector pQE-9.(Qiagen, Inc., Chatsworth, Calif.). This plasmid vector encodesantibiotic resistance (Ampr), a bacterial origin of replication (ori),an IPTG-regulatable promoter/operator (P/O), a ribosome binding site(RBS), a 6-histidine tag (6-His), and restriction enzyme cloning sites.

The pQE-9 vector is digested with BamHI and XbaI and the amplifiedfragment is ligated into the pQE-9 vector maintaining the reading frameinitiated at the bacterial RBS. The ligation mixture is then used totransform the E. coli strain M15/rep4 (Qiagen, Inc.) which containsmultiple copies of the plasmid pREP4, that expresses the lacI repressorand also confers kanamycin resistance (Kanr). Transformants areidentified by their ability to grow on LB plates andampicillin/kanamycin resistant colonies are selected. Plasmid DNA isisolated and confirmed by restriction analysis.

Clones containing the desired constructs are grown overnight (O/N) inliquid culture in LB media supplemented with both Amp (100 ug/ml) andKan (25 ug/ml). The O/N culture is used to inoculate a large culture ata ratio of 1:100 to 1:250. The cells are grown to an optical density 600(O.D.600) of between 0.4 and 0.6. IPTG(Isopropyl-B-D-thiogalactopyranoside) is then added to a finalconcentration of 1 mM. IPTG induces by inactivating the lacI repressor,clearing the P/0 leading to increased gene expression.

Cells are grown for an extra 3 to 4 hours. Cells are then harvested bycentrifugation (20 mins at 6000×g). The cell pellet is solubilized inthe chaotropic agent 6 Molar Guanidine HCl by stirring for 3-4 hours at4 degree C. The cell debris is removed by centrifugation, and thesupernatant containing the polypeptide is loaded onto anickel-nitrilo-tri-acetic acid (“Ni-NTA”) affinity resin column(available from QIAGEN, Inc., supra). Proteins with a 6×His tag bind tothe Ni-NTA resin with high affinity and can be purified in a simpleone-step procedure (for details see: The QIAexpressionist (1995) QIAGEN,Inc., supra).

Briefly, the supernatant is loaded onto the column in 6 M guanidine-HCl,pH 8, the column is first washed with 10 volumes of 6 M guanidine-HCl,pH 8, then washed with 10 volumes of 6 M guanidine-HCl pH 6, and finallythe polypeptide is eluted with 6 M guanidine-HCl, pH 5.

The purified protein is then renatured by dialyzing it againstphosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus200 mM NaCl. Alternatively, the protein can be successfully refoldedwhile immobilized on the Ni-NTA column. The recommended conditions areas follows: renature using a linear 6M-1M urea gradient in 500 mM NaCl,20% glycerol, 20 mM Tris/HCl pH 7.4, containing protease inhibitors. Therenaturation should be performed over a period of 1.5 hours or more.After renaturation the proteins are eluted by the addition of 250 mMimidazole. Imidazole is removed by a final dialyzing step against PBS or50 mM sodium acetate pH 6 buffer plus 200 mM NaCl. The purified proteinis stored at 4 degree C. or frozen at −80 degree C.

Example 14 Purification of a Polypeptide from an Inclusion Body

The following alternative method can be used to purify a polypeptideexpressed in E coli when it is present in the form of inclusion bodies.Unless otherwise specified, all of the following steps are conducted at4-10 degree C.

Upon completion of the production phase of the E. coli fermentation, thecell culture is cooled to 4-10 degree C. and the cells harvested bycontinuous centrifugation at 15,000 rpm (Heraeus Sepatech). On the basisof the expected yield of protein per unit weight of cell paste and theamount of purified protein required, an appropriate amount of cellpaste, by weight, is suspended in a buffer solution containing 100 mMTris, 50 mM EDTA, pH 7.4. The cells are dispersed to a homogeneoussuspension using a high shear mixer.

The cells are then lysed by passing the solution through amicrofluidizer (Microfluidics, Corp. or APV Gaulin, Inc.) twice at4000-6000 psi. The homogenate is then mixed with NaCl solution to afinal concentration of 0.5 M NaCl, followed by centrifugation at 7000×gfor 15 min. The resultant pellet is washed again using 0.5M NaCl, 100 mMTris, 50 mM EDTA, pH 7.4.

The resulting washed inclusion bodies are solubilized with 1.5 Mguanidine hydrochloride (GuHCl) for 2-4 hours. After 7000×gcentrifugation for 15 min., the pellet is discarded and the polypeptidecontaining supernatant is incubated at 4 degree C. overnight to allowfurther GuHCl extraction.

Following high speed centrifugation (30,000×g) to remove insolubleparticles, the GuHCl solubilized protein is refolded by quickly mixingthe GuHCl extract with 20 volumes of buffer containing 50 mM sodium, pH4.5, 150 mM NaCl, 2 mM EDTA by vigorous stirring. The refolded dilutedprotein solution is kept at 4 degree C. without mixing for 12 hoursprior to further purification steps.

To clarify the refolded polypeptide solution, a previously preparedtangential filtration unit equipped with 0.16 um membrane filter withappropriate surface area (e.g., Filtron), equilibrated with 40 mM sodiumacetate, pH 6.0 is employed. The filtered sample is loaded onto a cationexchange resin (e.g., Poros HS-50, Perceptive Biosystems). The column iswashed with 40 mM sodium acetate, pH 6.0 and eluted with 250 mM, 500 mM,1000 mM, and 1500 mM NaCl in the same buffer, in a stepwise manner. Theabsorbance at 280 nm of the effluent is continuously monitored.Fractions are collected and further analyzed by SDS-PAGE.

Fractions containing the polypeptide are then pooled and mixed with 4volumes of water. The diluted sample is then loaded onto a previouslyprepared set of tandem columns of strong anion (Poros HQ-50, PerceptiveBiosystems) and weak anion (Poros CM-20, Perceptive Biosystems) exchangeresins. The columns are equilibrated with 40 mM sodium acetate, pH 6.0.Both columns are washed with 40 mM sodium acetate, pH 6.0, 200 mM NaCl.The CM-20 column is then eluted using a 10 column volume linear gradientranging from 0.2 M NaCl, 50 mM sodium acetate, pH 6.0 to 1.0 M NaCl, 50mM sodium acetate, pH 6.5. Fractions are collected under constant A280monitoring of the effluent. Fractions containing the polypeptide(determined, for instance, by 16% SDS-PAGE) are then pooled.

The resultant polypeptide should exhibit greater than 95% purity afterthe above refolding and purification steps. No major contaminant bandsshould be observed from Coomassie blue stained 16% SDS-PAGE gel when 5ug of purified protein is loaded. The purified protein can also betested for endotoxin/LPS contamination, and typically the LPS content isless than 0.1 ng/ml according to LAL assays.

Example 15 Cloning and Expression of a Polypeptide in a BaculovirusExpression System

In this example, the plasmid shuttle vector pAc373 is used to insert apolynucleotide into a baculovirus to express a polypeptide. A typicalbaculovirus expression vector contains the strong polyhedrin promoter ofthe Autographa californica nuclear polyhedrosis virus (AcMNPV) followedby convenient restriction sites, which may include, for example BamHI,Xba I and Asp718. The polyadenylation site of the simian virus 40(“SV40”) is often used for efficient polyadenylation. For easy selectionof recombinant virus, the plasmid contains the beta-galactosidase genefrom E. coli under control of a weak Drosophila promoter in the sameorientation, followed by the polyadenylation signal of the polyhedringene. The inserted genes are flanked on both sides by viral sequencesfor cell-mediated homologous recombination with wild-type viral DNA togenerate a viable virus that express the cloned polynucleotide.

Many other baculovirus vectors can be used in place of the vector above,such as pVL941 and pAcIM1, as one skilled in the art would readilyappreciate, as long as the construct provides appropriately locatedsignals for transcription, translation, secretion and the like,including a signal peptide and an in-frame AUG as required. Such vectorsare described, for instance, in Luckow et al., Virology 170:31-39(1989).

A polynucleotide encoding a polypeptide of the present invention isamplified using PCR oligonucleotide primers corresponding to the 5′ and3′ ends of the DNA sequence, as outlined in Example 10, to synthesizeinsertion fragments. The primers used to amplify the cDNA insert shouldpreferably contain restriction sites at the 5′ end of the primers inorder to clone the amplified product into the expression vector.Specifically, the cDNA sequence contained in the deposited clone,including the AUG initiation codon and the naturally associated leadersequence identified elsewhere herein (if applicable), is amplified usingthe PCR protocol described in Example 10. If the naturally occurringsignal sequence is used to produce the protein, the vector used does notneed a second signal peptide. Alternatively, the vector can be modifiedto include a baculovirus leader sequence, using the standard methodsdescribed in Summers et al., “A Manual of Methods for BaculovirusVectors and Insect Cell Culture Procedures,” Texas AgriculturalExperimental Station Bulletin No. 1555 (1987).

The amplified fragment is isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment then is digested with appropriate restrictionenzymes and again purified on a 1% agarose gel.

The plasmid is digested with the corresponding restriction enzymes andoptionally, can be dephosphorylated using calf intestinal phosphatase,using routine procedures known in the art. The DNA is then isolated froma 1% agarose gel using a commercially available kit (“Geneclean” BIO 101Inc., La Jolla, Calif.).

The fragment and the dephosphorylated plasmid are ligated together withT4 DNA ligase. E. coli HB101 or other suitable E. coli hosts such asXL-1 Blue (Stratagene Cloning Systems, La Jolla, Calif.) cells aretransformed with the ligation mixture and spread on culture plates.Bacteria containing the plasmid are identified by digesting DNA fromindividual colonies and analyzing the digestion product by gelelectrophoresis. The sequence of the cloned fragment is confirmed by DNAsequencing.

Five ug of a plasmid containing the polynucleotide is co-transformedwith 1.0 ug of a commercially available linearized baculovirus DNA(“BaculoGold™ baculovirus DNA”, Pharmingen, San Diego, Calif.), usingthe lipofection method described by Felgner et al., Proc. Natl. Acad.Sci. USA 84:7413-7417 (1987). One ug of BaculoGold™ virus DNA and 5 ugof the plasmid are mixed in a sterile well of a microtiter platecontaining 50 ul of serum-free Grace's medium (Life Technologies Inc.,Gaithersburg, Md.). Afterwards, 10 ul Lipofectin plus 90 ul Grace'smedium are added, mixed and incubated for 15 minutes at roomtemperature. Then the transfection mixture is added drop-wise to Sf9insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with1 ml Grace's medium without serum. The plate is then incubated for 5hours at 27 degrees C. The transfection solution is then removed fromthe plate and 1 ml of Grace's insect medium supplemented with 10% fetalcalf serum is added. Cultivation is then continued at 27 degrees C. forfour days.

After four days the supernatant is collected and a plaque assay isperformed, as described by Summers and Smith, supra. An agarose gel with“Blue Gal” (Life Technologies Inc., Gaithersburg) is used to allow easyidentification and isolation of gal-expressing clones, which produceblue-stained plaques. (A detailed description of a “plaque assay” ofthis type can also be found in the user's guide for insect cell cultureand baculovirology distributed by Life Technologies Inc., Gaithersburg,page 9-10.) After appropriate incubation, blue stained plaques arepicked with the tip of a micropipettor (e.g., Eppendorf). The agarcontaining the recombinant viruses is then resuspended in amicrocentrifuge tube containing 200 ul of Grace's medium and thesuspension containing the recombinant baculovirus is used to infect Sf9cells seeded in 35 mm dishes. Four days later the supernatants of theseculture dishes are harvested and then they are stored at 4 degree C.

To verify the expression of the polypeptide, Sf9 cells are grown inGrace's medium supplemented with 10% heat-inactivated FBS. The cells areinfected with the recombinant baculovirus containing the polynucleotideat a multiplicity of infection (“MOI”) of about 2. If radiolabeledproteins are desired, 6 hours later the medium is removed and isreplaced with SF900 II medium minus methionine and cysteine (availablefrom Life Technologies Inc., Rockville, Md.). After 42 hours, 5 uCi of35S-methionine and 5 uCi 35S-cysteine (available from Amersham) areadded. The cells are further incubated for 16 hours and then areharvested by centrifugation. The proteins in the supernatant as well asthe intracellular proteins are analyzed by SDS-PAGE followed byautoradiography (if radiolabeled).

Microsequencing of the amino acid sequence of the amino terminus ofpurified protein may be used to determine the amino terminal sequence ofthe produced protein.

Example 16 Expression of a Polypeptide in Mammalian Cells

The polypeptide of the present invention can be expressed in a mammaliancell. A typical mammalian expression vector contains a promoter element,which mediates the initiation of transcription of mRNA, a protein codingsequence, and signals required for the termination of transcription andpolyadenylation of the transcript. Additional elements includeenhancers, Kozak sequences and intervening sequences flanked by donorand acceptor sites for RNA splicing. Highly efficient transcription isachieved with the early and late promoters from SV40, the long terminalrepeats (LTRs) from Retroviruses, e.g., RSV, HTLVI, HIVI and the earlypromoter of the cytomegalovirus (CMV). However, cellular elements canalso be used (e.g., the human actin promoter).

Suitable expression vectors for use in practicing the present inventioninclude, for example, vectors such as pSVL and pMSG (Pharmacia, Uppsala,Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146), pBC12MI (ATCC67109), pCMVSport 2.0, and pCMVSport 3.0. Mammalian host cells thatcould be used include, human Hela, 293, H9 and Jurkat cells, mouseNIH3T3 and C127 cells, Cos 1, Cos 7 and CV1, quail QC1-3 cells, mouse Lcells and Chinese hamster ovary (CHO) cells.

Alternatively, the polypeptide can be expressed in stable cell linescontaining the polynucleotide integrated into a chromosome. Theco-transformation with a selectable marker such as dhfr, gpt, neomycin,hygromycin allows the identification and isolation of the transformedcells.

The transformed gene can also be amplified to express large amounts ofthe encoded protein. The DHFR (dihydrofolate reductase) marker is usefulin developing cell lines that carry several hundred or even severalthousand copies of the gene of interest. (See, e.g., Alt, F. W., et al.,J. Biol. Chem. 253:1357-1370 (1978); Hamlin, J. L. and Ma, C., Biochem.et Biophys. Acta, 1097:107-143 (1990); Page, M. J. and Sydenham, M. A.,Biotechnology 9:64-68 (1991).) Another useful selection marker is theenzyme glutamine synthase (GS) (Murphy et al., Biochem J. 227:277-279(1991); Bebbington et al., Bio/Technology 10:169-175 (1992). Using thesemarkers, the mammalian cells are grown in selective medium and the cellswith the highest resistance are selected. These cell lines contain theamplified gene(s) integrated into a chromosome. Chinese hamster ovary(CHO) and NSO cells are often used for the production of proteins.

A polynucleotide of the present invention is amplified according to theprotocol outlined in herein. If the naturally occurring signal sequenceis used to produce the protein, the vector does not need a second signalpeptide. Alternatively, if the naturally occurring signal sequence isnot used, the vector can be modified to include a heterologous signalsequence. (See, e.g., WO 96/34891.) The amplified fragment is isolatedfrom a 1% agarose gel using a commercially available kit (“Geneclean,”BIO 101 Inc., La Jolla, Calif.). The fragment then is digested withappropriate restriction enzymes and again purified on a 1% agarose gel.

The amplified fragment is then digested with the same restriction enzymeand purified on a 1% agarose gel. The isolated fragment and thedephosphorylated vector are then ligated with T4 DNA ligase. E. coliHB101 or XL-1 Blue cells are then transformed and bacteria areidentified that contain the fragment inserted into plasmid pC6 using,for instance, restriction enzyme analysis.

Chinese hamster ovary cells lacking an active DHFR gene is used fortransformation. Five μg of an expression plasmid is cotransformed with0.5 μg of the plasmid pSVneo using lipofectin (Feigner et al., supra).The plasmid pSV2-neo contains a dominant selectable marker, the neo genefrom Tn5 encoding an enzyme that confers resistance to a group ofantibiotics including G418. The cells are seeded in alpha minus MEMsupplemented with 1 mg/ml G418. After 2 days, the cells are trypsinizedand seeded in hybridoma cloning plates (Greiner, Germany) in alpha minusMEM supplemented with 10, 25, or 50 ng/ml of methotrexate plus 1 mg/mlG418. After about 10-14 days single clones are trypsinized and thenseeded in 6-well petri dishes or 10 ml flasks using differentconcentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM).Clones growing at the highest concentrations of methotrexate are thentransferred to new 6-well plates containing even higher concentrationsof methotrexate (1 uM, 2 uM, 5 uM, 10 mM, 20 mM). The same procedure isrepeated until clones are obtained which grow at a concentration of100-200 uM. Expression of the desired gene product is analyzed, forinstance, by SDS-PAGE and Western blot or by reversed phase HPLCanalysis.

Example 17 Protein Fusions

The polypeptides of the present invention are preferably fused to otherproteins. These fusion proteins can be used for a variety ofapplications. For example, fusion of the present polypeptides toHis-tag, HA-tag, protein A, IgG domains, and maltose binding proteinfacilitates purification. (See Example described herein; see also EP A394,827; Traunecker, et al., Nature 331:84-86 (1988).) Similarly, fusionto IgG-1, IgG-3, and albumin increases the half-life time in vivo.Nuclear localization signals fused to the polypeptides of the presentinvention can target the protein to a specific subcellular localization,while covalent heterodimer or homodimers can increase or decrease theactivity of a fusion protein. Fusion proteins can also create chimericmolecules having more than one function. Finally, fusion proteins canincrease solubility and/or stability of the fused protein compared tothe non-fused protein. All of the types of fusion proteins describedabove can be made by modifying the following protocol, which outlinesthe fusion of a polypeptide to an IgG molecule.

Briefly, the human Fc portion of the IgG molecule can be PCR amplified,using primers that span the 5′ and 3′ ends of the sequence describedbelow. These primers also should have convenient restriction enzymesites that will facilitate cloning into an expression vector, preferablya mammalian expression vector. Note that the polynucleotide is clonedwithout a stop codon, otherwise a fusion protein will not be produced.

The naturally occurring signal sequence may be used to produce theprotein (if applicable). Alternatively, if the naturally occurringsignal sequence is not used, the vector can be modified to include aheterologous signal sequence. (See, e.g., WO 96/34891 and/or U.S. Pat.No. 6,066,781, supra.)

Human IgG Fc Region:

(SEQ ID NO:76)      GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGGTGGTGGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAACCCGCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAAGAACGAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAAGTACAAGACCACGGCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTGCGACGGCCGCGACTCTAGAGGAT

Example 18 Production of an Antibody from a Polypeptide

The antibodies of the present invention can be prepared by a variety ofmethods. (See, Current Protocols, Chapter 2.) As one example of suchmethods, cells expressing a polypeptide of the present invention areadministered to an animal to induce the production of sera containingpolyclonal antibodies. In a preferred method, a preparation of theprotein is prepared and purified to render it substantially free ofnatural contaminants. Such a preparation is then introduced into ananimal in order to produce polyclonal antisera of greater specificactivity.

In the most preferred method, the antibodies of the present inventionare monoclonal antibodies (or protein binding fragments thereof). Suchmonoclonal antibodies can be prepared using hybridoma technology.(Köhler et al., Nature 256:495 (1975); Köhler et al., Eur. J. Immunol.6:511 (1976); Köhler et al., Eur. J. Immunol. 6:292 (1976); Hammerlinget al., in: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y.,pp. 563-681 (1981).) In general, such procedures involve immunizing ananimal (preferably a mouse) with polypeptide or, more preferably, with apolypeptide-expressing cell. Such cells may be cultured in any suitabletissue culture medium; however, it is preferable to culture cells inEarle's modified Eagle's medium supplemented with 10% fetal bovine serum(inactivated at about 56 degrees C.), and supplemented with about 10 g/lof nonessential amino acids, about 1,000 U/ml of penicillin, and about100 ug/ml of streptomycin.

The splenocytes of such mice are extracted and fused with a suitablemyeloma cell line. Any suitable myeloma cell line may be employed inaccordance with the present invention; however, it is preferable toemploy the parent myeloma cell line (SP20), available from the ATCC.After fusion, the resulting hybridoma cells are selectively maintainedin HAT medium, and then cloned by limiting dilution as described byWands et al. (Gastroenterology 80:225-232 (1981).) The hybridoma cellsobtained through such a selection are then assayed to identify cloneswhich secrete antibodies capable of binding the polypeptide.

Alternatively, additional antibodies capable of binding to thepolypeptide can be produced in a two-step procedure using anti-idiotypicantibodies. Such a method makes use of the fact that antibodies arethemselves antigens, and therefore, it is possible to obtain an antibodythat binds to a second antibody. In accordance with this method, proteinspecific antibodies are used to immunize an animal, preferably a mouse.The splenocytes of such an animal are then used to produce hybridomacells, and the hybridoma cells are screened to identify clones thatproduce an antibody whose ability to bind to the protein-specificantibody can be blocked by the polypeptide. Such antibodies compriseanti-idiotypic antibodies to the protein-specific antibody and can beused to immunize an animal to induce formation of furtherprotein-specific antibodies.

It will be appreciated that Fab and F(ab′)2 and other fragments of theantibodies of the present invention may be used according to the methodsdisclosed herein. Such fragments are typically produced by proteolyticcleavage, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)2 fragments). Alternatively, protein-bindingfragments can be produced through the application of recombinant DNAtechnology or through synthetic chemistry.

For in vivo use of antibodies in humans, it may be preferable to use“humanized” chimeric monoclonal antibodies. Such antibodies can beproduced using genetic constructs derived from hybridoma cells producingthe monoclonal antibodies described above. Methods for producingchimeric antibodies are known in the art. (See, for review, Morrison,Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabillyet al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrisonet al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al.,Nature 314:268 (1985).)

Moreover, in another preferred method, the antibodies directed againstthe polypeptides of the present invention may be produced in plants.Specific methods are disclosed in U.S. Pat. Nos. 5,959,177, and6,080,560, which are hereby incorporated in their entirety herein. Themethods not only describe methods of expressing antibodies, but also themeans of assembling foreign multimeric proteins in plants (i.e.,antibodies, etc,), and the subsequent secretion of such antibodies fromthe plant.

Example 19 Regulation of Protein Expression Via Controlled Aggregationin the Endoplasmic Reticulum

As described more particularly herein, proteins regulate diversecellular processes in higher organisms, ranging from rapid metabolicchanges to growth and differentiation. Increased production of specificproteins could be used to prevent certain diseases and/or diseasestates. Thus, the ability to modulate the expression of specificproteins in an organism would provide significant benefits.

Numerous methods have been developed to date for introducing foreigngenes, either under the control of an inducible, constitutively active,or endogenous promoter, into organisms. Of particular interest are theinducible promoters (see, M. Gossen, et al., Proc. Natl. Acad. Sci.USA., 89:5547 (1992); Y. Wang, et al., Proc. Natl. Acad. Sci. USA,91:8180 (1994), D. No., et al., Proc. Natl. Acad. Sci. USA, 93:3346(1996); and V. M. Rivera, et al., Nature Med, 2:1028 (1996); in additionto additional examples disclosed elsewhere herein). In one example, thegene for erthropoietin (Epo) was transferred into mice and primatesunder the control of a small molecule inducer for expression (e.g.,tetracycline or rapamycin) (see, D. Bohl, et al., Blood, 92:1512,(1998); K. G. Rendahl, et al., Nat. Biotech, 16:757, (1998); V. M.Rivera, et al., Proc. Natl. Acad. Sci. USA, 96:8657 (1999); and X. Ye etal., Science, 283:88 (1999). Although such systems enable efficientinduction of the gene of interest in the organism upon addition of theinducing agent (i.e., tetracycline, rapamycin, etc.,), the levels ofexpression tend to peak at 24 hours and trail off to background levelsafter 4 to 14 days. Thus, controlled transient expression is virtuallyimpossible using these systems, though such control would be desirable.

A new alternative method of controlling gene expression levels of aprotein from a transgene (i.e., includes stable and transienttransformants) has recently been elucidated (V. M. Rivera., et al.,Science, 287:826-830, (2000)). This method does not control geneexpression at the level of the mRNA like the aforementioned systems.Rather, the system controls the level of protein in an active secretedform. In the absence of the inducing agent, the protein aggregates inthe ER and is not secreted. However, addition of the inducing agentresults in dis-aggregation of the protein and the subsequent secretionfrom the ER. Such a system affords low basal secretion, rapid, highlevel secretion in the presence of the inducing agent, and rapidcessation of secretion upon removal of the inducing agent. In fact,protein secretion reached a maximum level within 30 minutes ofinduction, and a rapid cessation of secretion within 1 hour of removingthe inducing agent. The method is also applicable for controlling thelevel of production for membrane proteins.

Detailed methods are presented in V. M. Rivera., et al., Science,287:826-830, (2000)), briefly:

Fusion protein constructs are created using polynucleotide sequences ofthe present invention with one or more copies (preferably at least 2, 3,4, or more) of a conditional aggregation domain (CAD) a domain thatinteracts with itself in a ligand-reversible manner (i.e., in thepresence of an inducing agent) using molecular biology methods known inthe art and discussed elsewhere herein. The CAD domain may be the mutantdomain isolated from the human FKBP12 (Phe³⁶ to Met) protein (asdisclosed in V. M. Rivera., et al., Science, 287:826-830, (2000), oralternatively other proteins having domains with similarligand-reversible, self-aggregation properties. As a principle of designthe fusion protein vector would contain a furin cleavage sequenceoperably linked between the polynucleotides of the present invention andthe CAD domains. Such a cleavage site would enable the proteolyticcleavage of the CAD domains from the polypeptide of the presentinvention subsequent to secretion from the ER and upon entry into thetrans-Golgi (J. B. Denault, et al., FEBS Lett., 379:113, (1996)).Alternatively, the skilled artisan would recognize that any proteolyticcleavage sequence could be substituted for the furin sequence providedthe substituted sequence is cleavable either endogenously (e.g., thefurin sequence) or exogenously (e.g., post secretion, post purification,post production, etc.). The preferred sequence of each feature of thefusion protein construct, from the 5′ to 3′ direction with each featurebeing operably linked to the other, would be a promoter, signalsequence, “X” number of (CAD)x domains, the furin sequence (or otherproteolytic sequence), and the coding sequence of the polypeptide of thepresent invention. The artisan would appreciate that the promotor andsignal sequence, independent from the other, could be either theendogenous promotor or signal sequence of a polypeptide of the presentinvention, or alternatively, could be a heterologous signal sequence andpromotor.

The specific methods described herein for controlling protein secretionlevels through controlled ER aggregation are not meant to be limitingare would be generally applicable to any of the polynucleotides andpolypeptides of the present invention, including variants, homologues,orthologs, and fragments therein.

Example 20 Alteration of Protein Glycosylation Sites to EnhanceCharacteristics of Polypeptides of the Invention

Many eukaryotic cell surface and proteins are post-translationallyprocessed to incorporate N-linked and O-linked carbohydrates (Kornfeldand Kornfeld (1985) Annu. Rev. Biochem. 54:631-64; Rademacher et al.,(1988) Annu. Rev. Biochem. 57:785-838). Protein glycosylation is thoughtto serve a variety of functions including: augmentation of proteinfolding, inhibition of protein aggregation, regulation of intracellulartrafficking to organelles, increasing resistance to proteolysis,modulation of protein antigenicity, and mediation of intercellularadhesion (Fieldler and Simons (1995) Cell, 81:309-312; Helenius (1994)Mol. Biol. Of the Cell 5:253-265; Olden et al., (1978) Cell, 13:461-473;Caton et al., (1982) Cell, 37:417-427; Alexamnder and Elder (1984),Science, 226:1328-1330; and Flack et al., (1994), J. Biol. Chem.,269:14015-14020). In higher organisms, the nature and extent ofglycosylation can markedly affect the circulating half-life andbio-availability of proteins by mechanisms involving receptor mediateduptake and clearance (Ashwell and Morrell, (1974), Adv. Enzymol.,41:99-128; Ashwell and Harford (1982), Ann. Rev. Biochem., 51:531-54).Receptor systems have been identified that are thought to play a majorrole in the clearance of serum proteins through recognition of variouscarbohydrate structures on the glycoproteins (Stockert (1995), Physiol.Rev., 75:591-609; Kery et al., (1992), Arch. Biochem. Biophys.,298:49-55). Thus, production strategies resulting in incompleteattachment of terminal sialic acid residues might provide a means ofshortening the bioavailability and half-life of glycoproteins.Conversely, expression strategies resulting in saturation of terminalsialic acid attachment sites might lengthen protein bioavailability andhalf-life.

In the development of recombinant glycoproteins for use aspharmaceutical products, for example, it has been speculated that thepharmacodynamics of recombinant proteins can be modulated by theaddition or deletion of glycosylation sites from a glycoproteins primarystructure (Berman and Lasky (1985a) Trends in Biotechnol., 3:51-53).However, studies have reported that the deletion of N-linkedglycosylation sites often impairs intracellular transport and results inthe intracellular accumulation of glycosylation site variants (Machamerand Rose (1988), J. Biol. Chem., 263:5955-5960; Gallagher et al.,(1992), J. Virology., 66:7136-7145; Collier et al., (1993), Biochem.,32:7818-7823; Claffey et al., (1995) Biochemica et Biophysica Acta,1246:1-9; Dube et al., (1988), J. Biol. Chem. 263:17516-17521). Whileglycosylation site variants of proteins can be expressedintracellularly, it has proved difficult to recover useful quantitiesfrom growth conditioned cell culture medium.

Moreover, it is unclear to what extent a glycosylation site in onespecies will be recognized by another species glycosylation machinery.Due to the importance of glycosylation in protein metabolism,particularly the secretion and/or expression of the protein, whether aglycosylation signal is recognized may profoundly determine a proteinsability to be expressed, either endogenously or recombinately, inanother organism (i.e., expressing a human protein in E. coli, yeast, orviral organisms; or an E. coli, yeast, or viral protein in human, etc.).Thus, it may be desirable to add, delete, or modify a glycosylationsite, and possibly add a glycosylation site of one species to a proteinof another species to improve the proteins functional, bioprocesspurification, and/or structural characteristics (e.g., a polypeptide ofthe present invention).

A number of methods may be employed to identify the location ofglycosylation sites within a protein. One preferred method is to run thetranslated protein sequence through the PROSITE computer program (SwissInstitute of Bioinformatics). Once identified, the sites could besystematically deleted, or impaired, at the level of the DNA usingmutagenesis methodology known in the art and available to the skilledartisan, Preferably using PCR-directed mutagenesis (See Maniatis,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, ColdSpring, N.Y. (1982)). Similarly, glycosylation sites could be added, ormodified at the level of the DNA using similar methods, preferably PCRmethods (See, Maniatis, supra). The results of modifying theglycosylation sites for a particular protein (e.g., solubility,secretion potential, activity, aggregation, proteolytic resistance,etc.) could then be analyzed using methods know in the art.

The skilled artisan would acknowledge the existence of other computeralgorithms capable of predicting the location of glycosylation siteswithin a protein. For example, the Motif computer program (GeneticsComputer Group suite of programs) provides this function, as well.

Example 21 Method of Enhancing the Biological Activity/FunctionalCharacteristics of Invention Through Molecular Evolution

Although many of the most biologically active proteins known are highlyeffective for their specified function in an organism, they oftenpossess characteristics that make them undesirable for transgenic,therapeutic, and/or industrial applications. Among these traits, a shortphysiological half-life is the most prominent problem, and is presenteither at the level of the protein, or the level of the proteins mRNA.The ability to extend the half-life, for example, would be particularlyimportant for a proteins use in gene therapy, transgenic animalproduction, the bioprocess production and purification of the protein,and use of the protein as a chemical modulator among others. Therefore,there is a need to identify novel variants of isolated proteinspossessing characteristics which enhance their application as atherapeutic for treating diseases of animal origin, in addition to theproteins applicability to common industrial and pharmaceuticalapplications.

Thus, one aspect of the present invention relates to the ability toenhance specific characteristics of invention through directed molecularevolution. Such an enhancement may, in a non-limiting example, benefitthe inventions utility as an essential component in a kit, theinventions physical attributes such as its solubility, structure, orcodon optimization, the inventions specific biological activity,including any associated enzymatic activity, the proteins enzymekinetics, the proteins Ki, Kcat, Km, Vmax, Kd, protein-protein activity,protein-DNA binding activity, antagonist/inhibitory activity (includingdirect or indirect interaction), agonist activity (including direct orindirect interaction), the proteins antigenicity (e.g., where it wouldbe desirable to either increase or decrease the antigenic potential ofthe protein), the immunogenicity of the protein, the ability of theprotein to form dimers, trimers, or multimers with either itself orother proteins, the antigenic efficacy of the invention, including itssubsequent use a preventative treatment for disease or disease states,or as an effector for targeting diseased genes. Moreover, the ability toenhance specific characteristics of a protein may also be applicable tochanging the characterized activity of an enzyme to an activitycompletely unrelated to its initially characterized activity. Otherdesirable enhancements of the invention would be specific to eachindividual protein, and would thus be well known in the art andcontemplated by the present invention.

For example, an engineered phosphatase may be constitutively active.Alternatively, an engineered phosphatase may be constitutively active inthe absence of ligand binding. In yet another example, an engineeredphosphatase may be capable of being activated with less than all of theregulatory factors and/or conditions typically required for phosphataseactivation (e.g., ligand binding, phosphorylation, conformationalchanges, etc.). Alternatively, an engineered phosphatase may havealtered substrate specificity. Such phosphatases would be useful inscreens to identify phosphatase modulators, among other uses describedherein.

Directed evolution is comprised of several steps. The first step is toestablish a library of variants for the gene or protein of interest. Themost important step is to then select for those variants that entail theactivity you wish to identify. The design of the screen is essentialsince your screen should be selective enough to eliminate non-usefulvariants, but not so stringent as to eliminate all variants. The laststep is then to repeat the above steps using the best variant from theprevious screen. Each successive cycle, can then be tailored asnecessary, such as increasing the stringency of the screen, for example.

Over the years, there have been a number of methods developed tointroduce mutations into macromolecules. Some of these methods include,random mutagenesis, “error-prone” PCR, chemical mutagenesis,site-directed mutagenesis, and other methods well known in the art (fora comprehensive listing of current mutagenesis methods, see Maniatis,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, ColdSpring, N.Y. (1982)). Typically, such methods have been used, forexample, as tools for identifying the core functional region(s) of aprotein or the function of specific domains of a protein (if amulti-domain protein). However, such methods have more recently beenapplied to the identification of macromolecule variants with specific orenhanced characteristics.

Random mutagenesis has been the most widely recognized method to date.Typically, this has been carried out either through the use of“error-prone” PCR (as described in Moore, J., et al, NatureBiotechnology 14:458, (1996), or through the application of randomizedsynthetic oligonucleotides corresponding to specific regions of interest(as described by Derbyshire, K. M. et al, Gene, 46:145-152, (1986), andHill, D E, et al, Methods Enzymol., 55:559-568, (1987). Both approacheshave limits to the level of mutagenesis that can be obtained. However,either approach enables the investigator to effectively control the rateof mutagenesis. This is particularly important considering the fact thatmutations beneficial to the activity of the enzyme are fairly rare. Infact, using too high a level of mutagenesis may counter or inhibit thedesired benefit of a useful mutation.

While both of the aforementioned methods are effective for creatingrandomized pools of macromolecule variants, a third method, termed “DNAShuffling”, or “sexual PCR” (WPC, Stemmer, PNAS, 91:10747, (1994)) hasrecently been elucidated. DNA shuffling has also been referred to as“directed molecular evolution”, “exon-shuffling”, “directed enzymeevolution”, “in vitro evolution”, and “artificial evolution”. Suchreference terms are known in the art and are encompassed by theinvention. This new, preferred, method apparently overcomes thelimitations of the previous methods in that it not only propagatespositive traits, but simultaneously eliminates negative traits in theresulting progeny.

DNA shuffling accomplishes this task by combining the principal of invitro recombination, along with the method of “error-prone” PCR. Ineffect, you begin with a randomly digested pool of small fragments ofyour gene, created by Dnase I digestion, and then introduce said randomfragments into an “error-prone” PCR assembly reaction. During the PCRreaction, the randomly sized DNA fragments not only hybridize to theircognate strand, but also may hybridize to other DNA fragmentscorresponding to different regions of the polynucleotide ofinterest—regions not typically accessible via hybridization of theentire polynucleotide. Moreover, since the PCR assembly reactionutilizes “error-prone” PCR reaction conditions, random mutations areintroduced during the DNA synthesis step of the PCR reaction for all ofthe fragments—further diversifying the potential hybridization sitesduring the annealing step of the reaction.

A variety of reaction conditions could be utilized to carry-out the DNAshuffling reaction. However, specific reaction conditions for DNAshuffling are provided, for example, in PNAS, 91:10747, (1994). Briefly:

Prepare the DNA substrate to be subjected to the DNA shuffling reaction.Preparation may be in the form of simply purifying the DNA fromcontaminating cellular material, chemicals, buffers, oligonucleotideprimers, deoxynucleotides, RNAs, etc., and may entail the use of DNApurification kits as those provided by Qiagen, Inc., or by the Promega,Corp., for example.

Once the DNA substrate has been purified, it would be subjected to DnaseI digestion. About 2-4 ug of the DNA substrate(s) would be digested with0.0015 units of Dnase I (Sigma) per ul in 100 ul of 50 mM Tris-HCL, pH7.4/1 mM MgCl2 for 10-20 min. at room temperature. The resultingfragments of 10-50 bp could then be purified by running them through a2% low-melting point agarose gel by electrophoresis onto DE81ion-exchange paper (Whatmann) or could be purified using Microconconcentrators (Amicon) of the appropriate molecular weight cutoff, orcould use oligonucleotide purification columns (Qiagen), in addition toother methods known in the art. If using DE81 ion-exchange paper, the10-50 bp fragments could be eluted from said paper using 1M NaCl,followed by ethanol precipitation.

The resulting purified fragments would then be subjected to a PCRassembly reaction by re-suspension in a PCR mixture containing: 2 mM ofeach dNTP, 2.2 mM MgCl2, 50 mM KCl, 10 mM Tris.HCL, pH 9.0, and 0.1%Triton X-100, at a final fragment concentration of 10-30 ng/ul. Noprimers are added at this point. Taq DNA polymerase (Promega) would beused at 2.5 units per 100 ul of reaction mixture. A PCR program of 94 Cfor 60 s; 94 C for 30 s, 50-55 C for 30 s, and 72 C for 30 s using 30-45cycles, followed by 72 C for 5 min using an MJ Research (Cambridge,Mass.) PTC-150 thermocycler. After the assembly reaction is completed, a1:40 dilution of the resulting primerless product would then beintroduced into a PCR mixture (using the same buffer mixture used forthe assembly reaction) containing 0.8 um of each primer and subjectingthis mixture to 15 cycles of PCR (using 94 C for 30 s, 50 C for 30 s,and 72 C for 30 s). The referred primers would be primers correspondingto the nucleic acid sequences of the polynucleotide(s) utilized in theshuffling reaction. Said primers could consist of modified nucleic acidbase pairs using methods known in the art and referred to else whereherein, or could contain additional sequences (i.e., for addingrestriction sites, mutating specific base-pairs, etc.).

The resulting shuffled, assembled, and amplified product can be purifiedusing methods well known in the art (e.g., Qiagen PCR purification kits)and then subsequently cloned using appropriate restriction enzymes.

Although a number of variations of DNA shuffling have been published todate, such variations would be obvious to the skilled artisan and areencompassed by the invention. The DNA shuffling method can also betailored to the desired level of mutagenesis using the methods describedby Zhao, et al. (Nucl Acid Res., 25(6):1307-1308, (1997).

As described above, once the randomized pool has been created, it canthen be subjected to a specific screen to identify the variantpossessing the desired characteristic(s). Once the variant has beenidentified, DNA corresponding to the variant could then be used as theDNA substrate for initiating another round of DNA shuffling. This cycleof shuffling, selecting the optimized variant of interest, and thenre-shuffling, can be repeated until the ultimate variant is obtained.Examples of model screens applied to identify variants created using DNAshuffling technology may be found in the following publications: J. C.,Moore, et al., J. Mol. Biol., 272:336-347, (1997), F. R., Cross, et al.,Mol. Cell. Biol., 18:2923-2931, (1998), and A. Crameri., et al., Nat.Biotech., 15:436-438, (1997).

DNA shuffling has several advantages. First, it makes use of beneficialmutations. When combined with screening, DNA shuffling allows thediscovery of the best mutational combinations and does not assume thatthe best combination contains all the mutations in a population.Secondly, recombination occurs simultaneously with point mutagenesis. Aneffect of forcing DNA polymerase to synthesize full-length genes fromthe small fragment DNA pool is a background mutagenesis rate. Incombination with a stringent selection method, enzymatic activity hasbeen evolved up to 16000 fold increase over the wild-type form of theenzyme. In essence, the background mutagenesis yielded the geneticvariability on which recombination acted to enhance the activity.

A third feature of recombination is that it can be used to removedeleterious mutations. As discussed above, during the process of therandomization, for every one beneficial mutation, there may be at leastone or more neutral or inhibitory mutations. Such mutations can beremoved by including in the assembly reaction an excess of the wild-typerandom-size fragments, in addition to the random-size fragments of theselected mutant from the previous selection. During the next selection,some of the most active variants of thepolynucleotide/polypeptide/enzyme, should have lost the inhibitorymutations.

Finally, recombination enables parallel processing. This represents asignificant advantage since there are likely multiple characteristicsthat would make a protein more desirable (e.g. solubility, activity,etc.). Since it is increasingly difficult to screen for more than onedesirable trait at a time, other methods of molecular evolution tend tobe inhibitory. However, using recombination, it would be possible tocombine the randomized fragments of the best representative variants forthe various traits, and then select for multiple properties at once.

DNA shuffling can also be applied to the polynucleotides andpolypeptides of the present invention to decrease their immunogenicityin a specified host. For example, a particular variant of the presentinvention may be created and isolated using DNA shuffling technology.Such a variant may have all of the desired characteristics, though maybe highly immunogenic in a host due to its novel intrinsic structure.Specifically, the desired characteristic may cause the polypeptide tohave a non-native structure which could no longer be recognized as a“self” molecule, but rather as a “foreign”, and thus activate a hostimmune response directed against the novel variant. Such a limitationcan be overcome, for example, by including a copy of the gene sequencefor a xenobiotic ortholog of the native protein in with the genesequence of the novel variant gene in one or more cycles of DNAshuffling. The molar ratio of the ortholog and novel variant DNAs couldbe varied accordingly. Ideally, the resulting hybrid variant identifiedwould contain at least some of the coding sequence which enabled thexenobiotic protein to evade the host immune system, and additionally,the coding sequence of the original novel variant that provided thedesired characteristics.

Likewise, the invention encompasses the application of DNA shufflingtechnology to the evolution of polynucleotides and polypeptides of theinvention, wherein one or more cycles of DNA shuffling include, inaddition to the gene template DNA, oligonucleotides coding for knownallelic sequences, optimized codon sequences, known variant sequences,known polynucleotide polymorphism sequences, known ortholog sequences,known homologue sequences, additional homologous sequences, additionalnon-homologous sequences, sequences from another species, and any numberand combination of the above.

In addition to the described methods above, there are a number ofrelated methods that may also be applicable, or desirable in certaincases. Representative among these are the methods discussed in PCTapplications WO 98/31700, and WO 98/32845, which are hereby incorporatedby reference. Furthermore, related methods can also be applied to thepolynucleotide sequences of the present invention in order to evolveinvention for creating ideal variants for use in gene therapy, proteinengineering, evolution of whole cells containing the variant, or in theevolution of entire enzyme pathways containing polynucleotides of theinvention as described in PCT applications WO 98/13485, WO 98/13487, WO98/27230, WO 98/31837, and Crameri, A., et al., Nat. Biotech.,15:436-438, (1997), respectively.

Additional methods of applying “DNA Shuffling” technology to thepolynucleotides and polypeptides of the present invention, includingtheir proposed applications, may be found in U.S. Pat. No. 5,605,793;PCT Application No. WO 95/22625; PCT Application No. WO 97/20078; PCTApplication No. WO 97/35966; and PCT Application No. WO 98/42832; PCTApplication No. WO 00/09727 specifically provides methods for applyingDNA shuffling to the identification of herbicide selective crops whichcould be applied to the polynucleotides and polypeptides of the presentinvention; additionally, PCT Application No. WO 00/12680 providesmethods and compositions for generating, modifying, adapting, andoptimizing polynucleotide sequences that confer detectable phenotypicproperties on plant species; each of the above are hereby incorporatedin their entirety herein for all purposes.

Example 22 Method of Determining Alterations in a Gene Corresponding toa Polynucleotide

RNA isolated from entire families or individual patients presenting witha phenotype of interest (such as a disease) is be isolated. cDNA is thengenerated from these RNA samples using protocols known in the art. (See,Sambrook.) The cDNA is then used as a template for PCR, employingprimers surrounding regions of interest in SEQ ID NO:X. Suggested PCRconditions consist of 35 cycles at 95 degrees C. for 30 seconds; 60-120seconds at 52-58 degrees C.; and 60-120 seconds at 70 degrees C., usingbuffer solutions described in Sidransky et al., Science 252:706 (1991).

PCR products are then sequenced using primers labeled at their 5′ endwith T4 polynucleotide kinase, employing SequiTherm Polymerase.(Epicentre Technologies). The intron-exon borders of selected exons isalso determined and genomic PCR products analyzed to confirm theresults. PCR products harboring suspected mutations is then cloned andsequenced to validate the results of the direct sequencing.

PCR products is cloned into T-tailed vectors as described in Holton etal., Nucleic Acids Research, 19:1156 (1991) and sequenced with T7polymerase (United States Biochemical). Affected individuals areidentified by mutations not present in unaffected individuals.

Genomic rearrangements are also observed as a method of determiningalterations in a gene corresponding to a polynucleotide. Genomic clonesisolated according to Example 2 are nick-translated withdigoxigenindeoxy-uridine 5′-triphosphate (Boehringer Manheim), and FISHperformed as described in Johnson et al., Methods Cell Biol. 35:73-99(1991). Hybridization with the labeled probe is carried out using a vastexcess of human cot-1 DNA for specific hybridization to thecorresponding genomic locus.

Chromosomes are counterstained with 4,6-diamino-2-phenylidole andpropidium iodide, producing a combination of C- and R-bands. Alignedimages for precise mapping are obtained using a triple-band filter set(Chroma Technology, Brattleboro, Vt.) in combination with a cooledcharge-coupled device camera (Photometrics, Tucson, Ariz.) and variableexcitation wavelength filters. (Johnson et al., Genet. Anal. Tech.Appl., 8:75 (1991).) Image collection, analysis and chromosomalfractional length measurements are performed using the ISee GraphicalProgram System. (Inovision Corporation, Durham, N.C.) Chromosomealterations of the genomic region hybridized by the probe are identifiedas insertions, deletions, and translocations. These alterations are usedas a diagnostic marker for an associated disease.

Example 23 Method of Detecting Abnormal Levels of a Polypeptide in aBiological Sample

A polypeptide of the present invention can be detected in a biologicalsample, and if an increased or decreased level of the polypeptide isdetected, this polypeptide is a marker for a particular phenotype.Methods of detection are numerous, and thus, it is understood that oneskilled in the art can modify the following assay to fit theirparticular needs.

For example, antibody-sandwich ELISAs are used to detect polypeptides ina sample, preferably a biological sample. Wells of a microtiter plateare coated with specific antibodies, at a final concentration of 0.2 to10 ug/ml. The antibodies are either monoclonal or polyclonal and areproduced by the method described elsewhere herein. The wells are blockedso that non-specific binding of the polypeptide to the well is reduced.

The coated wells are then incubated for >2 hours at RT with a samplecontaining the polypeptide. Preferably, serial dilutions of the sampleshould be used to validate results. The plates are then washed threetimes with deionized or distilled water to remove unbounded polypeptide.

Next, 50 ul of specific antibody-alkaline phosphatase conjugate, at aconcentration of 25-400 ng, is added and incubated for 2 hours at roomtemperature. The plates are again washed three times with deionized ordistilled water to remove unbounded conjugate.

Add 75 ul of 4-methylumbelliferyl phosphate (MUP) or p-nitrophenylphosphate (NPP) substrate solution to each well and incubate 1 hour atroom temperature. Measure the reaction by a microtiter plate reader.Prepare a standard curve, using serial dilutions of a control sample,and plot polypeptide concentration on the X-axis (log scale) andfluorescence or absorbance of the Y-axis (linear scale). Interpolate theconcentration of the polypeptide in the sample using the standard curve.

Example 24 Formulation

The invention also provides methods of treatment and/or preventiondiseases, disorders, and/or conditions (such as, for example, any one ormore of the diseases or disorders disclosed herein) by administration toa subject of an effective amount of a Therapeutic. By therapeutic ismeant a polynucleotides or polypeptides of the invention (includingfragments and variants), agonists or antagonists thereof, and/orantibodies thereto, in combination with a pharmaceutically acceptablecarrier type (e.g., a sterile carrier).

The Therapeutic will be formulated and dosed in a fashion consistentwith good medical practice, taking into account the clinical conditionof the individual patient (especially the side effects of treatment withthe Therapeutic alone), the site of delivery, the method ofadministration, the scheduling of administration, and other factorsknown to practitioners. The “effective amount” for purposes herein isthus determined by such considerations.

As a general proposition, the total pharmaceutically effective amount ofthe Therapeutic administered parenterally per dose will be in the rangeof about 1 ug/kg/day to 10 mg/kg/day of patient body weight, although,as noted above, this will be subject to therapeutic discretion. Morepreferably, this dose is at least 0.01 mg/kg/day, and most preferablyfor humans between about 0.01 and 1 mg/kg/day for the hormone. If givencontinuously, the Therapeutic is typically administered at a dose rateof about 1 ug/kg/hour to about 50 ug/kg/hour, either by 1-4 injectionsper day or by continuous subcutaneous infusions, for example, using amini-pump. An intravenous bag solution may also be employed. The lengthof treatment needed to observe changes and the interval followingtreatment for responses to occur appears to vary depending on thedesired effect.

Therapeutics can be administered orally, rectally, parenterally,intracisternally, intravaginally, intraperitoneally, topically (as bypowders, ointments, gels, drops or transdermal patch), bucally, or as anoral or nasal spray. “Pharmaceutically acceptable carrier” refers to anon-toxic solid, semisolid or liquid filler, diluent, encapsulatingmaterial or formulation auxiliary of any. The term “parenteral” as usedherein refers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrasternal, subcutaneous andintraarticular injection and infusion.

Therapeutics of the invention are also suitably administered bysustained-release systems. Suitable examples of sustained-releaseTherapeutics are administered orally, rectally, parenterally,intracisternally, intravaginally, intraperitoneally, topically (as bypowders, ointments, gels, drops or transdermal patch), bucally, or as anoral or nasal spray. “Pharmaceutically acceptable carrier” refers to anon-toxic solid, semisolid or liquid filler, diluent, encapsulatingmaterial or formulation auxiliary of any type. The term “parenteral” asused herein refers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrasternal, subcutaneous andintraarticular injection and infusion.

Therapeutics of the invention may also be suitably administered bysustained-release systems. Suitable examples of sustained-releaseTherapeutics include suitable polymeric materials (such as, for example,semi-permeable polymer matrices in the form of shaped articles, e.g.,films, or microcapsules), suitable hydrophobic materials (for example asan emulsion in an acceptable oil) or ion exchange resins, and sparinglysoluble derivatives (such as, for example, a sparingly soluble salt).

Sustained-release matrices include polylactides (U.S. Pat. No.3,773,919, EP 58,481), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (Sidman et al., Biopolymers 22:547-556 (1983)),poly (2-hydroxyethyl methacrylate) (Langer et al., J. Biomed. Mater.Res. 15:167-277 (1981), and Langer, Chem. Tech. 12:98-105 (1982)),ethylene vinyl acetate (Langer et al., Id.) orpoly-D-(−)-3-hydroxybutyric acid (EP 133,988).

Sustained-release Therapeutics also include liposomally entrappedTherapeutics of the invention (see, generally, Langer, Science249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy ofInfectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss,New York, pp. 317-327 and 353-365 (1989)). Liposomes containing theTherapeutic are prepared by methods known per se: DE 3,218,121; Epsteinet al., Proc. Natl. Acad. Sci. (USA) 82:3688-3692 (1985); Hwang et al.,Proc. Natl. Acad. Sci. (USA) 77:4030-4034 (1980); EP 52,322; EP 36,676;EP 88,046; EP 143,949; EP 142,641; Japanese Pat. Appl. 83-118008; U.S.Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily, theliposomes are of the small (about 200-800 Angstroms) unilamellar type inwhich the lipid content is greater than about 30 mol. percentcholesterol, the selected proportion being adjusted for the optimalTherapeutic.

In yet an additional embodiment, the Therapeutics of the invention aredelivered by way of a pump (see Langer, supra; Sefton, CRC Crit. Ref.Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980);Saudek et al., N. Engl. J. Med. 321:574 (1989)).

Other controlled release systems are discussed in the review by Langer(Science 249:1527-1533 (1990)).

For parenteral administration, in one embodiment, the Therapeutic isformulated generally by mixing it at the desired degree of purity, in aunit dosage injectable form (solution, suspension, or emulsion), with apharmaceutically acceptable carrier, i.e., one that is non-toxic torecipients at the dosages and concentrations employed and is compatiblewith other ingredients of the formulation. For example, the formulationpreferably does not include oxidizing agents and other compounds thatare known to be deleterious to the Therapeutic.

Generally, the formulations are prepared by contacting the Therapeuticuniformly and intimately with liquid carriers or finely divided solidcarriers or both. Then, if necessary, the product is shaped into thedesired formulation. Preferably the carrier is a parenteral carrier,more preferably a solution that is isotonic with the blood of therecipient. Examples of such carrier vehicles include water, saline,Ringer's solution, and dextrose solution. Non-aqueous vehicles such asfixed oils and ethyl oleate are also useful herein, as well asliposomes.

The carrier suitably contains minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, mannose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; counterions such as sodium; and/or nonionicsurfactants such as polysorbates, poloxamers, or PEG.

The Therapeutic will typically be formulated in such vehicles at aconcentration of about 0.1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml, ata pH of about 3 to 8. It will be understood that the use of certain ofthe foregoing excipients, carriers, or stabilizers will result in theformation of polypeptide salts.

Any pharmaceutical used for therapeutic administration can be sterile.Sterility is readily accomplished by filtration through sterilefiltration membranes (e.g., 0.2 micron membranes). Therapeuticsgenerally are placed into a container having a sterile access port, forexample, an intravenous solution bag or vial having a stopper pierceableby a hypodermic injection needle.

Therapeutics ordinarily will be stored in unit or multi-dose containers,for example, sealed ampoules or vials, as an aqueous solution or as alyophilized formulation for reconstitution. As an example of alyophilized formulation, 10-ml vials are filled with 5 ml ofsterile-filtered 1% (w/v) aqueous Therapeutic solution, and theresulting mixture is lyophilized. The infusion solution is prepared byreconstituting the lyophilized Therapeutic using bacteriostaticWater-for-Injection.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of theTherapeutics of the invention. Associated with such container(s) can bea notice in the form prescribed by a governmental agency regulating themanufacture, use or sale of pharmaceuticals or biological products,which notice reflects approval by the agency of manufacture, use or salefor human administration. In addition, the Therapeutics may be employedin conjunction with other therapeutic compounds.

The Therapeutics of the invention may be administered alone or incombination with adjuvants. Adjuvants that may be administered with theTherapeutics of the invention include, but are not limited to, alum,alum plus deoxycholate (ImmunoAg), MTP-PE (Biocine Corp.), QS21(Genentech, Inc.), BCG, and MPL. In a specific embodiment, Therapeuticsof the invention are administered in combination with alum. In anotherspecific embodiment, Therapeutics of the invention are administered incombination with QS-21. Further adjuvants that may be administered withthe Therapeutics of the invention include, but are not limited to,Monophosphoryl lipid immunomodulator, AdjuVax 100a, QS-21, QS-18,CRL1005, Aluminum salts, MF-59, and Virosomal adjuvant technology.Vaccines that may be administered with the Therapeutics of the inventioninclude, but are not limited to, vaccines directed toward protectionagainst MMR (measles, mumps, rubella), polio, varicella,tetanus/diptheria, hepatitis A, hepatitis B, haemophilus influenzae B,whooping cough, pneumonia, influenza, Lyme's Disease, rotavirus,cholera, yellow fever, Japanese encephalitis, poliomyelitis, rabies,typhoid fever, and pertussis. Combinations may be administered eitherconcomitantly, e.g., as an admixture, separately but simultaneously orconcurrently; or sequentially. This includes presentations in which thecombined agents are administered together as a therapeutic mixture, andalso procedures in which the combined agents are administered separatelybut simultaneously, e.g., as through separate intravenous lines into thesame individual. Administration “in combination” further includes theseparate administration of one of the compounds or agents given first,followed by the second.

The Therapeutics of the invention may be administered alone or incombination with other therapeutic agents. Therapeutic agents that maybe administered in combination with the Therapeutics of the invention,include but not limited to, other members of the TNF family,chemotherapeutic agents, antibiotics, steroidal and non-steroidalanti-inflammatories, conventional immunotherapeutic agents, cytokinesand/or growth factors. Combinations may be administered eitherconcomitantly, e.g., as an admixture, separately but simultaneously orconcurrently; or sequentially. This includes presentations in which thecombined agents are administered together as a therapeutic mixture, andalso procedures in which the combined agents are administered separatelybut simultaneously, e.g., as through separate intravenous lines into thesame individual. Administration “in combination” further includes theseparate administration of one of the compounds or agents given first,followed by the second.

In one embodiment, the Therapeutics of the invention are administered incombination with members of the TNF family. TNF, TNF-related or TNF-likemolecules that may be administered with the Therapeutics of theinvention include, but are not limited to, soluble forms of TNF-alpha,lymphotoxin-alpha (LT-alpha, also known as TNF-beta), LT-beta (found incomplex heterotrimer LT-alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L,4-1BBL, DcR3, OX40L, TNF-gamma (International Publication No. WO96/14328), AIM-I (International Publication No. WO 97/33899),endokine-alpha (International Publication No. WO 98/07880), TR6(International Publication No. WO 98/30694), OPG, and neutrokine-alpha(International Publication No. WO 98/18921, OX40, and nerve growthfactor (NGF), and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB, TR2(International Publication No. WO 96/34095), DR3 (InternationalPublication No. WO 97/33904), DR4 (International Publication No. WO98/32856), TR5 (International Publication No. WO 98/30693), TR6(International Publication No. WO 98/30694), TR7 (InternationalPublication No. WO 98/41629), TRANK, TR9 (International Publication No.WO 98/56892), TR10 (International Publication No. WO 98/54202), 312C2(International Publication No. WO 98/06842), and TR12, and soluble formsCD154, CD70, and CD153.

In certain embodiments, Therapeutics of the invention are administeredin combination with antiretroviral agents, nucleoside reversetranscriptase inhibitors, non-nucleoside reverse transcriptaseinhibitors, and/or protease inhibitors. Nucleoside reverse transcriptaseinhibitors that may be administered in combination with the Therapeuticsof the invention, include, but are not limited to, RETROVIR((zidovudine/AZT), VIDEX( (didanosine/ddI), HIVID( (zalcitabine/ddC),ZERIT( (stavudine/d4T), EPIVIR( (lamivudine/3TC), and COMBIVIR((zidovudine/lamivudine). Non-nucleoside reverse transcriptase inhibitorsthat may be administered in combination with the Therapeutics of theinvention, include, but are not limited to, VIRAMUNE( (nevirapine),RESCRIPTOR( (delavirdine), and SUSTIVA( (efavirenz). Protease inhibitorsthat may be administered in combination with the Therapeutics of theinvention, include, but are not limited to, CRIXIVAN( (indinavir),NORVIR( (ritonavir), INVIRASE( (saquinavir), and VIRACEPT( (nelfinavir).In a specific embodiment, antiretroviral agents, nucleoside reversetranscriptase inhibitors, non-nucleoside reverse transcriptaseinhibitors, and/or protease inhibitors may be used in any combinationwith Therapeutics of the invention to treat AIDS and/or to prevent ortreat HIV infection.

In other embodiments, Therapeutics of the invention may be administeredin combination with anti-opportunistic infection agents.Anti-opportunistic agents that may be administered in combination withthe Therapeutics of the invention, include, but are not limited to,TRIMETHOPRIM-SULFAMETHOXAZOLE(, DAPSONE(, PENTAMIDINE(, ATOVAQUONE(,ISONIAZID(, RIFAMPIN(, PYRAZINAMIDE(, ETHAMBUTOL(, RIFABUTIN(,CLARITHROMYCIN(, AZITHROMYCIN(, GANCICLOVIR(, FOSCARNET(, CIDOFOVIR(,FLUCONAZOLE(, ITRACONAZOLE(, KETOCONAZOLE(, ACYCLOVIR(, FAMCICOLVIR(,PYRIMETHAMINE(, LEUCOVORIN(, NEUPOGEN( (filgrastim/G-CSF), and LEUKINE((sargramostim/GM-CSF). In a specific embodiment, Therapeutics of theinvention are used in any combination withTRIMETHOPRIM-SULFAMETHOXAZOLE(, DAPSONE(, PENTAMIDINE(, and/orATOVAQUONE( to prophylactically treat or prevent an opportunisticPneumocystis carinii pneumonia infection. In another specificembodiment, Therapeutics of the invention are used in any combinationwith ISONIAZID(, RIFAMPIN(, PYRAZINAMIDE(, and/or ETHAMBUTOL( toprophylactically treat or prevent an opportunistic Mycobacterium aviumcomplex infection. In another specific embodiment, Therapeutics of theinvention are used in any combination with RIFABUTIN(, CLARITHROMYCIN(,and/or AZITHROMYCIN( to prophylactically treat or prevent anopportunistic Mycobacterium tuberculosis infection. In another specificembodiment, Therapeutics of the invention are used in any combinationwith GANCICLOVIR(, FOSCARNET(, and/or CIDOFOVIR( to prophylacticallytreat or prevent an opportunistic cytomegalovirus infection. In anotherspecific embodiment, Therapeutics of the invention are used in anycombination with FLUCONAZOLE(, ITRACONAZOLE(, and/or KETOCONAZOLE( toprophylactically treat or prevent an opportunistic fungal infection. Inanother specific embodiment, Therapeutics of the invention are used inany combination with ACYCLOVIR( and/or FAMCICOLVIR( to prophylacticallytreat or prevent an opportunistic herpes simplex virus type I and/ortype II infection. In another specific embodiment, Therapeutics of theinvention are used in any combination with PYRIMETHAMINE( and/orLEUCOVORIN( to prophylactically treat or prevent an opportunisticToxoplasma gondii infection. In another specific embodiment,Therapeutics of the invention are used in any combination withLEUCOVORIN( and/or NEUPOGEN( to prophylactically treat or prevent anopportunistic bacterial infection.

In a further embodiment, the Therapeutics of the invention areadministered in combination with an antiviral agent. Antiviral agentsthat may be administered with the Therapeutics of the invention include,but are not limited to, acyclovir, ribavirin, amantadine, andremantidine.

In a further embodiment, the Therapeutics of the invention areadministered in combination with an antibiotic agent. Antibiotic agentsthat may be administered with the Therapeutics of the invention include,but are not limited to, amoxicillin, beta-lactamases, aminoglycosides,beta-lactam (glycopeptide), beta-lactamases, Clindamycin,chloramphenicol, cephalosporins, ciprofloxacin, ciprofloxacin,erythromycin, fluoroquinolones, macrolides, metronidazole, penicillins,quinolones, rifampin, streptomycin, sulfonamide, tetracyclines,trimethoprim, trimethoprim-sulfamthoxazole, and vancomycin.

Conventional nonspecific immunosuppressive agents, that may beadministered in combination with the Therapeutics of the inventioninclude, but are not limited to, steroids, cyclosporine, cyclosporineanalogs, cyclophosphamide methylprednisone, prednisone, azathioprine,FK-506, 15-deoxyspergualin, and other immunosuppressive agents that actby suppressing the function of responding T cells.

In specific embodiments, Therapeutics of the invention are administeredin combination with immunosuppressants. Immunosuppressants preparationsthat may be administered with the Therapeutics of the invention include,but are not limited to, ORTHOCLONE( (OKT3), SANDIMMUNE(/NEORAL(/SANGDYA((cyclosporin), PROGRAF( (tacrolimus), CELLCEPT( (mycophenolate),Azathioprine, glucorticosteroids, and RAPAMUNE( (sirolimus). In aspecific embodiment, immunosuppressants may be used to prevent rejectionof organ or bone marrow transplantation.

In an additional embodiment, Therapeutics of the invention areadministered alone or in combination with one or more intravenous immuneglobulin preparations. Intravenous immune globulin preparations that maybe administered with the Therapeutics of the invention include, but notlimited to, GAMMAR(, IVEEGAM(, SANDOGLOBULIN(, GAMMAGARD S/D(, andGAMIMUNE(. In a specific embodiment, Therapeutics of the invention areadministered in combination with intravenous immune globulinpreparations in transplantation therapy (e.g., bone marrow transplant).

In an additional embodiment, the Therapeutics of the invention areadministered alone or in combination with an anti-inflammatory agent.Anti-inflammatory agents that may be administered with the Therapeuticsof the invention include, but are not limited to, glucocorticoids andthe nonsteroidal anti-inflammatories, aminoarylcarboxylic acidderivatives, arylacetic acid derivatives, arylbutyric acid derivatives,arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles,pyrazolones, salicylic acid derivatives, thiazinecarboxamides,e-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyricacid, amixetrine, bendazac, benzydamine, bucolome, difenpiramide,ditazol, emorfazone, guaiazulene, nabumetone, nimesulide, orgotein,oxaceprol, paranyline, perisoxal, pifoxime, proquazone, proxazole, andtenidap.

In another embodiment, compositions of the invention are administered incombination with a chemotherapeutic agent. Chemotherapeutic agents thatmay be administered with the Therapeutics of the invention include, butare not limited to, antibiotic derivatives (e.g., doxorubicin,bleomycin, daunorubicin, and dactinomycin); antiestrogens (e.g.,tamoxifen); antimetabolites (e.g., fluorouracil, 5-FU, methotrexate,floxuridine, interferon alpha-2b, glutamic acid, plicamycin,mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g., carmustine,BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide,estramustine, hydroxyurea, procarbazine, mitomycin, busulfan,cis-platin, and vincristine sulfate); hormones (e.g.,medroxyprogesterone, estramustine phosphate sodium, ethinyl estradiol,estradiol, megestrol acetate, methyltestosterone, diethylstilbestroldiphosphate, chlorotrianisene, and testolactone); nitrogen mustardderivatives (e.g., mephalen, chorambucil, mechlorethamine (nitrogenmustard) and thiotepa); steroids and combinations (e.g., bethamethasonesodium phosphate); and others (e.g., dicarbazine, asparaginase,mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).

In a specific embodiment, formulations of the present invention mayfurther comprise antagonists of P-glycoprotein (also referred to as themultiresistance protein, or PGP), including antagonists of its encodingpolynucleotides (e.g., antisense oligonucleotides, ribozymes,zinc-finger proteins, etc.). P-glycoprotein is well known for decreasingthe efficacy of various drug administrations due to its ability toexport intracellular levels of absorbed drug to the cell exterior. Whilethis activity has been particularly pronounced in cancer cells inresponse to the administration of chemotherapy regimens, a variety ofother cell types and the administration of other drug classes have beennoted (e.g., T-cells and anti-HIV drugs). In fact, certain mutations inthe PGP gene significantly reduces PGP function, making it less able toforce drugs out of cells. People who have two versions of the mutatedgene—one inherited from each parent—have more than four times less PGPthan those with two normal versions of the gene. People may also haveone normal gene and one mutated one. Certain ethnic populations haveincreased incidence of such PGP mutations. Among individuals from Ghana,Kenya, the Sudan, as well as African Americans, frequency of the normalgene ranged from 73% to 84%. In contrast, the frequency was 34% to 59%among British whites, Portuguese, Southwest Asian, Chinese, Filipino andSaudi populations. As a result, certain ethnic populations may requireincreased administration of PGP antagonist in the formulation of thepresent invention to arrive at the an efficacious dose of thetherapeutic (e.g., those from African descent). Conversely, certainethnic populations, particularly those having increased frequency of themutated PGP (e.g., of Caucasian descent, or non-African descent) mayrequire less pharmaceutical compositions in the formulation due to aneffective increase in efficacy of such compositions as a result of theincreased effective absorption (e.g., less PGP activity) of saidcomposition.

Moreover, in another specific embodiment, formulations of the presentinvention may further comprise antagonists of OATP2 (also referred to asthe multiresistance protein, or MRP2), including antagonists of itsencoding polynucleotides (e.g., antisense oligonucleotides, ribozymes,zinc-finger proteins, etc.). The invention also further comprises anyadditional antagonists known to inhibit proteins thought to beattributable to a multidrug resistant phenotype in proliferating cells.

In a specific embodiment, Therapeutics of the invention are administeredin combination with CHOP (cyclophosphamide, doxorubicin, vincristine,and prednisone) or any combination of the components of CHOP. In anotherembodiment, Therapeutics of the invention are administered incombination with Rituximab. In a further embodiment, Therapeutics of theinvention are administered with Rituxmab and CHOP, or Rituxmab and anycombination of the components of CHOP.

In an additional embodiment, the Therapeutics of the invention areadministered in combination with cytokines. Cytokines that may beadministered with the Therapeutics of the invention include, but are notlimited to, IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13, IL-15,anti-CD40, CD40L, IFN-gamma and TNF-alpha. In another embodiment,Therapeutics of the invention may be administered with any interleukin,including, but not limited to, IL-1alpha, IL-1beta, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15,IL-16, IL-17, IL-18, IL-19, IL-20, and IL-21.

In an additional embodiment, the Therapeutics of the invention areadministered in combination with angiogenic proteins. Angiogenicproteins that may be administered with the Therapeutics of the inventioninclude, but are not limited to, Glioma Derived Growth Factor (GDGF), asdisclosed in European Patent Number EP-399816; Platelet Derived GrowthFactor-A (PDGF-A), as disclosed in European Patent Number EP-682110;Platelet Derived Growth Factor-B (PDGF-B), as disclosed in EuropeanPatent Number EP-282317; Placental Growth Factor (PlGF), as disclosed inInternational Publication Number WO 92/06194; Placental Growth Factor-2(PlGF-2), as disclosed in Hauser et al., Gorwth Factors, 4:259-268(1993); Vascular Endothelial Growth Factor (VEGF), as disclosed inInternational Publication Number WO 90/13649; Vascular EndothelialGrowth Factor-A (VEGF-A), as disclosed in European Patent NumberEP-506477; Vascular Endothelial Growth Factor-2 (VEGF-2), as disclosedin International Publication Number WO 96/39515; Vascular EndothelialGrowth Factor B (VEGF-3); Vascular Endothelial Growth Factor B-186(VEGF-B186), as disclosed in International Publication Number WO96/26736; Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed inInternational Publication Number WO 98/02543; Vascular EndothelialGrowth Factor-D (VEGF-D), as disclosed in International PublicationNumber WO 98/07832; and Vascular Endothelial Growth Factor-E (VEGF-E),as disclosed in German Patent Number DE19639601. The above mentionedreferences are incorporated herein by reference herein.

In an additional embodiment, the Therapeutics of the invention areadministered in combination with hematopoietic growth factors.Hematopoietic growth factors that may be administered with theTherapeutics of the invention include, but are not limited to, LEUKINE((SARGRAMOSTIM( ) and NEUPOGEN( (FILGRASTIM( ).

In an additional embodiment, the Therapeutics of the invention areadministered in combination with Fibroblast Growth Factors. FibroblastGrowth Factors that may be administered with the Therapeutics of theinvention include, but are not limited to, FGF-1, FGF-2, FGF-3, FGF-4,FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13,FGF-14, and FGF-15.

In additional embodiments, the Therapeutics of the invention areadministered in combination with other therapeutic or prophylacticregimens, such as, for example, radiation therapy.

Example 25 Method of Treating Decreased Levels of the Polypeptide

The present invention relates to a method for treating an individual inneed of an increased level of a polypeptide of the invention in the bodycomprising administering to such an individual a composition comprisinga therapeutically effective amount of an agonist of the invention(including polypeptides of the invention). Moreover, it will beappreciated that conditions caused by a decrease in the standard ornormal expression level of a secreted protein in an individual can betreated by administering the polypeptide of the present invention,preferably in the secreted form. Thus, the invention also provides amethod of treatment of an individual in need of an increased level ofthe polypeptide comprising administering to such an individual aTherapeutic comprising an amount of the polypeptide to increase theactivity level of the polypeptide in such an individual.

For example, a patient with decreased levels of a polypeptide receives adaily dose 0.1-100 ug/kg of the polypeptide for six consecutive days.Preferably, the polypeptide is in the secreted form. The exact detailsof the dosing scheme, based on administration and formulation, areprovided herein.

Example 26 Method of Treating Increased Levels of the Polypeptide

The present invention also relates to a method of treating an individualin need of a decreased level of a polypeptide of the invention in thebody comprising administering to such an individual a compositioncomprising a therapeutically effective amount of an antagonist of theinvention (including polypeptides and antibodies of the invention).

In one example, antisense technology is used to inhibit production of apolypeptide of the present invention. This technology is one example ofa method of decreasing levels of a polypeptide, preferably a secretedform, due to a variety of etiologies, such as cancer. For example, apatient diagnosed with abnormally increased levels of a polypeptide isadministered intravenously antisense polynucleotides at 0.5, 1.0, 1.5,2.0 and 3.0 mg/kg day for 21 days. This treatment is repeated after a7-day rest period if the treatment was well tolerated. The formulationof the antisense polynucleotide is provided herein.

Example 27 Method of Treatment Using Gene Therapy—Ex Vivo

One method of gene therapy transplants fibroblasts, which are capable ofexpressing a polypeptide, onto a patient. Generally, fibroblasts areobtained from a subject by skin biopsy. The resulting tissue is placedin tissue-culture medium and separated into small pieces. Small chunksof the tissue are placed on a wet surface of a tissue culture flask,approximately ten pieces are placed in each flask. The flask is turnedupside down, closed tight and left at room temperature over night. After24 hours at room temperature, the flask is inverted and the chunks oftissue remain fixed to the bottom of the flask and fresh media (e.g.,Ham's F12 media, with 10% FBS, penicillin and streptomycin) is added.The flasks are then incubated at 37 degree C. for approximately oneweek.

At this time, fresh media is added and subsequently changed everyseveral days. After an additional two weeks in culture, a monolayer offibroblasts emerge. The monolayer is trypsinized and scaled into largerflasks.

pMV-7 (Kirschmeier, P. T. et al., DNA, 7:219-25 (1988)), flanked by thelong terminal repeats of the Moloney murine sarcoma virus, is digestedwith EcoRI and HindIII and subsequently treated with calf intestinalphosphatase. The linear vector is fractionated on agarose gel andpurified, using glass beads.

The cDNA encoding a polypeptide of the present invention can beamplified using PCR primers which correspond to the 5′ and 3′ endsequences respectively as set forth in Example 10 using primers andhaving appropriate restriction sites and initiation/stop codons, ifnecessary. Preferably, the 5′ primer contains an EcoRI site and the 3′primer includes a HindIII site. Equal quantities of the Moloney murinesarcoma virus linear backbone and the amplified EcoRI and HindIIIfragment are added together, in the presence of T4 DNA ligase. Theresulting mixture is maintained under conditions appropriate forligation of the two fragments. The ligation mixture is then used totransform bacteria HB101, which are then plated onto agar containingkanamycin for the purpose of confirming that the vector has the gene ofinterest properly inserted.

The amphotropic pA317 or GP+am12 packaging cells are grown in tissueculture to confluent density in Dulbecco's Modified Eagles Medium (DMEM)with 10% calf serum (CS), penicillin and streptomycin. The MSV vectorcontaining the gene is then added to the media and the packaging cellstransduced with the vector. The packaging cells now produce infectiousviral particles containing the gene (the packaging cells are nowreferred to as producer cells).

Fresh media is added to the transduced producer cells, and subsequently,the media is harvested from a 10 cm plate of confluent producer cells.The spent media, containing the infectious viral particles, is filteredthrough a millipore filter to remove detached producer cells and thismedia is then used to infect fibroblast cells. Media is removed from asub-confluent plate of fibroblasts and quickly replaced with the mediafrom the producer cells. This media is removed and replaced with freshmedia. If the titer of virus is high, then virtually all fibroblastswill be infected and no selection is required. If the titer is very low,then it is necessary to use a retroviral vector that has a selectablemarker, such as neo or his. Once the fibroblasts have been efficientlyinfected, the fibroblasts are analyzed to determine whether protein isproduced.

The engineered fibroblasts are then transplanted onto the host, eitheralone or after having been grown to confluence on cytodex 3 microcarrierbeads.

Example 28 Gene Therapy Using Endogenous Genes Corresponding toPolynucleotides of the Invention

Another method of gene therapy according to the present inventioninvolves operably associating the endogenous polynucleotide sequence ofthe invention with a promoter via homologous recombination as described,for example, in U.S. Pat. No. 5,641,670, issued Jun. 24, 1997;International Publication NO: WO 96/29411, published Sep. 26, 1996;International Publication NO: WO 94/12650, published Aug. 4, 1994;Koller et al., Proc. Natl. Acad. Sci. USA, 86:8932-8935 (1989); andZijlstra et al., Nature, 342:435438 (1989). This method involves theactivation of a gene which is present in the target cells, but which isnot expressed in the cells, or is expressed at a lower level thandesired.

Polynucleotide constructs are made which contain a promoter andtargeting sequences, which are homologous to the 5′ non-coding sequenceof endogenous polynucleotide sequence, flanking the promoter. Thetargeting sequence will be sufficiently near the 5′ end of thepolynucleotide sequence so the promoter will be operably linked to theendogenous sequence upon homologous recombination. The promoter and thetargeting sequences can be amplified using PCR. Preferably, theamplified promoter contains distinct restriction enzyme sites on the 5′and 3′ ends. Preferably, the 3′ end of the first targeting sequencecontains the same restriction enzyme site as the 5′ end of the amplifiedpromoter and the 5′ end of the second targeting sequence contains thesame restriction site as the 3′ end of the amplified promoter.

The amplified promoter and the amplified targeting sequences aredigested with the appropriate restriction enzymes and subsequentlytreated with calf intestinal phosphatase. The digested promoter anddigested targeting sequences are added together in the presence of T4DNA ligase. The resulting mixture is maintained under conditionsappropriate for ligation of the two fragments. The construct is sizefractionated on an agarose gel then purified by phenol extraction andethanol precipitation.

In this Example, the polynucleotide constructs are administered as nakedpolynucleotides via electroporation. However, the polynucleotideconstructs may also be administered with transfection-facilitatingagents, such as liposomes, viral sequences, viral particles,precipitating agents, etc. Such methods of delivery are known in theart.

Once the cells are transfected, homologous recombination will take placewhich results in the promoter being operably linked to the endogenouspolynucleotide sequence. This results in the expression ofpolynucleotide corresponding to the polynucleotide in the cell.Expression may be detected by immunological staining, or any othermethod known in the art.

Fibroblasts are obtained from a subject by skin biopsy. The resultingtissue is placed in DMEM+10% fetal calf serum. Exponentially growing orearly stationary phase fibroblasts are trypsinized and rinsed from theplastic surface with nutrient medium. An aliquot of the cell suspensionis removed for counting, and the remaining cells are subjected tocentrifugation. The supernatant is aspirated and the pellet isresuspended in 5 ml of electroporation buffer (20 mM HEPES pH 7.3, 137mM NaCl, 5 mM KCl, 0.7 mM Na2 HPO4, 6 mM dextrose). The cells arerecentrifuged, the supernatant aspirated, and the cells resuspended inelectroporation buffer containing 1 mg/ml acetylated bovine serumalbumin. The final cell suspension contains approximately 3×106cells/ml. Electroporation should be performed immediately followingresuspension.

Plasmid DNA is prepared according to standard techniques. For example,to construct a plasmid for targeting to the locus corresponding to thepolynucleotide of the invention, plasmid pUC18 (MBI Fermentas, Amherst,N.Y.) is digested with HindIII. The CMV promoter is amplified by PCRwith an XbaI site on the 5′ end and a BamHI site on the 3′end. Twonon-coding sequences are amplified via PCR: one non-coding sequence(fragment 1) is amplified with a HindIII site at the 5′ end and an Xbasite at the 3′end; the other non-coding sequence (fragment 2) isamplified with a BamHI site at the 5′end and a HindIII site at the3′end. The CMV promoter and the fragments (1 and 2) are digested withthe appropriate enzymes (CMV promoter—XbaI and BamHI; fragment 1—XbaI;fragment 2—BamHI) and ligated together. The resulting ligation productis digested with HindIII, and ligated with the HindIII-digested pUC18plasmid.

Plasmid DNA is added to a sterile cuvette with a 0.4 cm electrode gap(Bio-Rad). The final DNA concentration is generally at least 120 μg/ml.0.5 ml of the cell suspension (containing approximately 1.5.×106 cells)is then added to the cuvette, and the cell suspension and DNA solutionsare gently mixed. Electroporation is performed with a Gene-Pulserapparatus (Bio-Rad). Capacitance and voltage are set at 960 μF and250-300 V, respectively. As voltage increases, cell survival decreases,but the percentage of surviving cells that stably incorporate theintroduced DNA into their genome increases dramatically. Given theseparameters, a pulse time of approximately 14-20 mSec should be observed.

Electroporated cells are maintained at room temperature forapproximately 5 min, and the contents of the cuvette are then gentlyremoved with a sterile transfer pipette. The cells are added directly to10 ml of prewarmed nutrient media (DMEM with 15% calf serum) in a 10 cmdish and incubated at 37 degree C. The following day, the media isaspirated and replaced with 10 ml of fresh media and incubated for afurther 16-24 hours.

The engineered fibroblasts are then injected into the host, either aloneor after having been grown to confluence on cytodex 3 microcarrierbeads. The fibroblasts now produce the protein product. The fibroblastscan then be introduced into a patient as described above.

Example 29 Method of Treatment Using Gene Therapy—In Vivo

Another aspect of the present invention is using in vivo gene therapymethods to treat disorders, diseases and conditions. The gene therapymethod relates to the introduction of naked nucleic acid (DNA, RNA, andantisense DNA or RNA) sequences into an animal to increase or decreasethe expression of the polypeptide. The polynucleotide of the presentinvention may be operatively linked to a promoter or any other geneticelements necessary for the expression of the polypeptide by the targettissue. Such gene therapy and delivery techniques and methods are knownin the art, see, for example, WO90/11092, WO98/11779; U.S. Pat. Nos.5,693,622, 5,705,151, 5,580,859; Tabata et al., Cardiovasc. Res.35(3):470-479 (1997); Chao et al., Pharmacol. Res. 35(6):517-522 (1997);Wolff, Neuromuscul. Disord. 7(5):314-318 (1997); Schwartz et al., GeneTher. 3(5):405-411 (1996); Tsurumi et al., Circulation 94(12):3281-3290(1996) (incorporated herein by reference).

The polynucleotide constructs may be delivered by any method thatdelivers injectable materials to the cells of an animal, such as,injection into the interstitial space of tissues (heart, muscle, skin,lung, liver, intestine and the like). The polynucleotide constructs canbe delivered in a pharmaceutically acceptable liquid or aqueous carrier.

The term “naked” polynucleotide, DNA or RNA, refers to sequences thatare free from any delivery vehicle that acts to assist, promote, orfacilitate entry into the cell, including viral sequences, viralparticles, liposome formulations, lipofectin or precipitating agents andthe like. However, the polynucleotides of the present invention may alsobe delivered in liposome formulations (such as those taught in FelgnerP. L. et al. (1995) Ann. NY Acad. Sci. 772:126-139 and Abdallah B. etal. (1995) Biol. Cell 85(1):1-7) which can be prepared by methods wellknown to those skilled in the art.

The polynucleotide vector constructs used in the gene therapy method arepreferably constructs that will not integrate into the host genome norwill they contain sequences that allow for replication. Any strongpromoter known to those skilled in the art can be used for driving theexpression of DNA. Unlike other gene therapies techniques, one majoradvantage of introducing naked nucleic acid sequences into target cellsis the transitory nature of the polynucleotide synthesis in the cells.Studies have shown that non-replicating DNA sequences can be introducedinto cells to provide production of the desired polypeptide for periodsof up to six months.

The polynucleotide construct can be delivered to the interstitial spaceof tissues within the an animal, including of muscle, skin, brain, lung,liver, spleen, bone marrow, thymus, heart, lymph, blood, bone,cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis,ovary, uterus, rectum, nervous system, eye, gland, and connectivetissue. Interstitial space of the tissues comprises the intercellularfluid, mucopolysaccharide matrix among the reticular fibers of organtissues, elastic fibers in the walls of vessels or chambers, collagenfibers of fibrous tissues, or that same matrix within connective tissueensheathing muscle cells or in the lacunae of bone. It is similarly thespace occupied by the plasma of the circulation and the lymph fluid ofthe lymphatic channels. Delivery to the interstitial space of muscletissue is preferred for the reasons discussed below. They may beconveniently delivered by injection into the tissues comprising thesecells. They are preferably delivered to and expressed in persistent,non-dividing cells which are differentiated, although delivery andexpression may be achieved in non-differentiated or less completelydifferentiated cells, such as, for example, stem cells of blood or skinfibroblasts. In vivo muscle cells are particularly competent in theirability to take up and express polynucleotides.

For the naked polynucleotide injection, an effective dosage amount ofDNA or RNA will be in the range of from about 0.05 g/kg body weight toabout 50 mg/kg body weight. Preferably the dosage will be from about0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05 mg/kgto about 5 mg/kg. Of course, as the artisan of ordinary skill willappreciate, this dosage will vary according to the tissue site ofinjection. The appropriate and effective dosage of nucleic acid sequencecan readily be determined by those of ordinary skill in the art and maydepend on the condition being treated and the route of administration.The preferred route of administration is by the parenteral route ofinjection into the interstitial space of tissues. However, otherparenteral routes may also be used, such as, inhalation of an aerosolformulation particularly for delivery to lungs or bronchial tissues,throat or mucous membranes of the nose. In addition, nakedpolynucleotide constructs can be delivered to arteries duringangioplasty by the catheter used in the procedure.

The dose response effects of injected polynucleotide in muscle in vivois determined as follows. Suitable template DNA for production of mRNAcoding for polypeptide of the present invention is prepared inaccordance with a standard recombinant DNA methodology. The templateDNA, which may be either circular or linear, is either used as naked DNAor complexed with liposomes. The quadriceps muscles of mice are theninjected with various amounts of the template DNA.

Five to six week old female and male Balb/C mice are anesthetized byintraperitoneal injection with 0.3 ml of 2.5% Avertin. A 1.5 cm incisionis made on the anterior thigh, and the quadriceps muscle is directlyvisualized. The template DNA is injected in 0.1 ml of carrier in a 1 ccsyringe through a 27 gauge needle over one minute, approximately 0.5 cmfrom the distal insertion site of the muscle into the knee and about 0.2cm deep. A suture is placed over the injection site for futurelocalization, and the skin is closed with stainless steel clips.

After an appropriate incubation time (e.g., 7 days) muscle extracts areprepared by excising the entire quadriceps. Every fifth 15 umcross-section of the individual quadriceps muscles is histochemicallystained for protein expression. A time course for protein expression maybe done in a similar fashion except that quadriceps from different miceare harvested at different times. Persistence of DNA in muscle followinginjection may be determined by Southern blot analysis after preparingtotal cellular DNA and HIRT supernatants from injected and control mice.The results of the above experimentation in mice can be use toextrapolate proper dosages and other treatment parameters in humans andother animals using naked DNA.

Example 30 Transgenic Animals

The polypeptides of the invention can also be expressed in transgenicanimals. Animals of any species, including, but not limited to, mice,rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep,cows and non-human primates, e.g., baboons, monkeys, and chimpanzees maybe used to generate transgenic animals. In a specific embodiment,techniques described herein or otherwise known in the art, are used toexpress polypeptides of the invention in humans, as part of a genetherapy protocol.

Any technique known in the art may be used to introduce the transgene(i.e., polynucleotides of the invention) into animals to produce thefounder lines of transgenic animals. Such techniques include, but arenot limited to, pronuclear microinjection (Paterson et al., Appl.Microbiol. Biotechnol. 40:691-698 (1994); Carver et al., Biotechnology(NY) 11:1263-1270 (1993); Wright et al., Biotechnology (NY) 9:830-834(1991); and Hoppe et al., U.S. Pat. No. 4,873,191 (1989)); retrovirusmediated gene transfer into germ lines (Van der Putten et al., Proc.Natl. Acad. Sci., USA 82:6148-6152 (1985)), blastocysts or embryos; genetargeting in embryonic stem cells (Thompson et al., Cell 56:313-321(1989)); electroporation of cells or embryos (Lo, 1983, Mol Cell. Biol.3:1803-1814 (1983)); introduction of the polynucleotides of theinvention using a gene gun (see, e.g., Ulmer et al., Science 259:1745(1993); introducing nucleic acid constructs into embryonic pleuripotentstem cells and transferring the stem cells back into the blastocyst; andsperm-mediated gene transfer (Lavitrano et al., Cell 57:717-723 (1989);etc. For a review of such techniques, see Gordon, “Transgenic Animals,”Intl. Rev. Cytol. 115:171-229 (1989), which is incorporated by referenceherein in its entirety.

Any technique known in the art may be used to produce transgenic clonescontaining polynucleotides of the invention, for example, nucleartransfer into enucleated oocytes of nuclei from cultured embryonic,fetal, or adult cells induced to quiescence (Campell et al., Nature380:64-66 (1996); Wilmut et al., Nature 385:810-813 (1997)).

The present invention provides for transgenic animals that carry thetransgene in all their cells, as well as animals which carry thetransgene in some, but not all their cells, i.e., mosaic animals orchimeric. The transgene may be integrated as a single transgene or asmultiple copies such as in concatamers, e.g., head-to-head tandems orhead-to-tail tandems. The transgene may also be selectively introducedinto and activated in a particular cell type by following, for example,the teaching of Lasko et al. (Lasko et al., Proc. Natl. Acad. Sci. USA89:6232-6236 (1992)). The regulatory sequences required for such acell-type specific activation will depend upon the particular cell typeof interest, and will be apparent to those of skill in the art. When itis desired that the polynucleotide transgene be integrated into thechromosomal site of the endogenous gene, gene targeting is preferred.Briefly, when such a technique is to be utilized, vectors containingsome nucleotide sequences homologous to the endogenous gene are designedfor the purpose of integrating, via homologous recombination withchromosomal sequences, into and disrupting the function of thenucleotide sequence of the endogenous gene. The transgene may also beselectively introduced into a particular cell type, thus inactivatingthe endogenous gene in only that cell type, by following, for example,the teaching of Gu et al. (Gu et al., Science 265:103-106 (1994)). Theregulatory sequences required for such a cell-type specific inactivationwill depend upon the particular cell type of interest, and will beapparent to those of skill in the art.

Once transgenic animals have been generated, the expression of therecombinant gene may be assayed utilizing standard techniques. Initialscreening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to verify that integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques which include, but are not limited to, Northern blot analysisof tissue samples obtained from the animal, in situ hybridizationanalysis, and reverse transcriptase-PCR(RT-PCR). Samples of transgenicgene-expressing tissue may also be evaluated immunocytochemically orimmunohistochemically using antibodies specific for the transgeneproduct.

Once the founder animals are produced, they may be bred, inbred,outbred, or crossbred to produce colonies of the particular animal.Examples of such breeding strategies include, but are not limited to:outbreeding of founder animals with more than one integration site inorder to establish separate lines; inbreeding of separate lines in orderto produce compound transgenics that express the transgene at higherlevels because of the effects of additive expression of each transgene;crossing of heterozygous transgenic animals to produce animalshomozygous for a given integration site in order to both augmentexpression and eliminate the need for screening of animals by DNAanalysis; crossing of separate homozygous lines to produce compoundheterozygous or homozygous lines; and breeding to place the transgene ona distinct background that is appropriate for an experimental model ofinterest.

Transgenic animals of the invention have uses which include, but are notlimited to, animal model systems useful in elaborating the biologicalfunction of polypeptides of the present invention, studying diseases,disorders, and/or conditions associated with aberrant expression, and inscreening for compounds effective in ameliorating such diseases,disorders, and/or conditions.

Example 31 Knock-Out Animals

Endogenous gene expression can also be reduced by inactivating or“knocking out” the gene and/or its promoter using targeted homologousrecombination. (E.g., see Smithies et al., Nature 317:230-234 (1985);Thomas & Capecchi, Cell 51:503-512 (1987); Thompson et al., Cell5:313-321 (1989); each of which is incorporated by reference herein inits entirety). For example, a mutant, non-functional polynucleotide ofthe invention (or a completely unrelated DNA sequence) flanked by DNAhomologous to the endogenous polynucleotide sequence (either the codingregions or regulatory regions of the gene) can be used, with or withouta selectable marker and/or a negative selectable marker, to transfectcells that express polypeptides of the invention in vivo. In anotherembodiment, techniques known in the art are used to generate knockoutsin cells that contain, but do not express the gene of interest.Insertion of the DNA construct, via targeted homologous recombination,results in inactivation of the targeted gene. Such approaches areparticularly suited in research and agricultural fields wheremodifications to embryonic stem cells can be used to generate animaloffspring with an inactive targeted gene (e.g., see Thomas & Capecchi1987 and Thompson 1989, supra). However this approach can be routinelyadapted for use in humans provided the recombinant DNA constructs aredirectly administered or targeted to the required site in vivo usingappropriate viral vectors that will be apparent to those of skill in theart.

In further embodiments of the invention, cells that are geneticallyengineered to express the polypeptides of the invention, oralternatively, that are genetically engineered not to express thepolypeptides of the invention (e.g., knockouts) are administered to apatient in vivo. Such cells may be obtained from the patient (i.e.,animal, including human) or an MHC compatible donor and can include, butare not limited to fibroblasts, bone marrow cells, blood cells (e.g.,lymphocytes), adipocytes, muscle cells, endothelial cells etc. The cellsare genetically engineered in vitro using recombinant DNA techniques tointroduce the coding sequence of polypeptides of the invention into thecells, or alternatively, to disrupt the coding sequence and/orendogenous regulatory sequence associated with the polypeptides of theinvention, e.g., by transduction (using viral vectors, and preferablyvectors that integrate the transgene into the cell genome) ortransfection procedures, including, but not limited to, the use ofplasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc. Thecoding sequence of the polypeptides of the invention can be placed underthe control of a strong constitutive or inducible promoter orpromoter/enhancer to achieve expression, and preferably secretion, ofthe polypeptides of the invention. The engineered cells which expressand preferably secrete the polypeptides of the invention can beintroduced into the patient systemically, e.g., in the circulation, orintraperitoneally.

Alternatively, the cells can be incorporated into a matrix and implantedin the body, e.g., genetically engineered fibroblasts can be implantedas part of a skin graft; genetically engineered endothelial cells can beimplanted as part of a lymphatic or vascular graft. (See, for example,Anderson et al. U.S. Pat. No. 5,399,349; and Mulligan & Wilson, U.S.Pat. No. 5,460,959 each of which is incorporated by reference herein inits entirety).

When the cells to be administered are non-autologous or non-MHCcompatible cells, they can be administered using well known techniqueswhich prevent the development of a host immune response against theintroduced cells. For example, the cells may be introduced in anencapsulated form which, while allowing for an exchange of componentswith the immediate extracellular environment, does not allow theintroduced cells to be recognized by the host immune system.

Transgenic and “knock-out” animals of the invention have uses whichinclude, but are not limited to, animal model systems useful inelaborating the biological function of polypeptides of the presentinvention, studying diseases, disorders, and/or conditions associatedwith aberrant expression, and in screening for compounds effective inameliorating such diseases, disorders, and/or conditions.

Example 32 Production of an Antibody

a) Hybridoma Technology

The antibodies of the present invention can be prepared by a variety ofmethods. (See, Current Protocols, Chapter 2.) As one example of suchmethods, cells expressing human phosphatase are administered to ananimal to induce the production of sera containing polyclonalantibodies. In a preferred method, a preparation of human phosphataseprotein is prepared and purified to render it substantially free ofnatural contaminants. Such a preparation is then introduced into ananimal in order to produce polyclonal antisera of greater specificactivity.

Monoclonal antibodies specific for protein human phosphatase areprepared using hybridoma technology. (Kohler et al., Nature 256:495(1975); Kohler et al., Eur. J. Immunol. 6:511 (1976); Kohler et al.,Eur. J. Immunol. 6:292 (1976); Hammerling et al., in: MonoclonalAntibodies and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-681 (1981)).In general, an animal (preferably a mouse) is immunized with humanphosphatase polypeptide or, more preferably, with a secreted humanphosphatase polypeptide-expressing cell. Such polypeptide-expressingcells are cultured in any suitable tissue culture medium, preferably inEarle's modified Eagle's medium supplemented with 10% fetal bovine serum(inactivated at about 56° C.), and supplemented with about 10 g/l ofnonessential amino acids, about 1,000 U/ml of penicillin, and about 100μg/ml of streptomycin.

The splenocytes of such mice are extracted and fused with a suitablemyeloma cell line. Any suitable myeloma cell line may be employed inaccordance with the present invention; however, it is preferable toemploy the parent myeloma cell line (SP2O), available from the ATCC.After fusion, the resulting hybridoma cells are selectively maintainedin HAT medium, and then cloned by limiting dilution as described byWands et al. (Gastroenterology 80:225-232 (1981)). The hybridoma cellsobtained through such a selection are then assayed to identify cloneswhich secrete antibodies capable of binding the human phosphatasepolypeptide.

Alternatively, additional antibodies capable of binding to humanphosphatase polypeptide can be produced in a two-step procedure usinganti-idiotypic antibodies. Such a method makes use of the fact thatantibodies are themselves antigens, and therefore, it is possible toobtain an antibody that binds to a second antibody. In accordance withthis method, protein specific antibodies are used to immunize an animal,preferably a mouse. The splenocytes of such an animal are then used toproduce hybridoma cells, and the hybridoma cells are screened toidentify clones which produce an antibody whose ability to bind to thehuman phosphatase protein-specific antibody can be blocked by humanphosphatase. Such antibodies comprise anti-idiotypic antibodies to thehuman phosphatase protein-specific antibody and are used to immunize ananimal to induce formation of further human phosphatase protein-specificantibodies.

For in vivo use of antibodies in humans, an antibody is “humanized”.Such antibodies can be produced using genetic constructs derived fromhybridoma cells producing the monoclonal antibodies described above.Methods for producing chimeric and humanized antibodies are known in theart and are discussed herein. (See, for review, Morrison, Science229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabilly et al.,U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al.,EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO 8702671;Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature314:268 (1985).)

b) Isolation of Antibody Fragments Directed

Against Human Phosphatase from a Library of scFvs

Naturally occurring V-genes isolated from human PBLs are constructedinto a library of antibody fragments which contain reactivities againsthuman phosphatase to which the donor may or may not have been exposed(see e.g., U.S. Pat. No. 5,885,793 incorporated herein by reference inits entirety).

Rescue of the Library. A library of scFvs is constructed from the RNA ofhuman PBLs as described in PCT publication WO 92/01047. To rescue phagedisplaying antibody fragments, approximately 109 E. coli harboring thephagemid are used to inoculate 50 ml of 2xTY containing 1% glucose and100 μg/ml of ampicillin (2xTY-AMP-GLU) and grown to an O.D. of 0.8 withshaking. Five ml of this culture is used to inoculate 50 ml of2xTY-AMP-GLU, 2×108 TU of delta gene 3 helper (M13 delta gene III, seePCT publication WO 92/01047) are added and the culture incubated at 37°C. for 45 minutes without shaking and then at 37° C. for 45 minutes withshaking. The culture is centrifuged at 4000 r.p.m. for 10 min. and thepellet resuspended in 2 liters of 2xTY containing 100 μg/ml ampicillinand 50 ug/ml kanamycin and grown overnight. Phage are prepared asdescribed in PCT publication WO 92/01047.

M13 delta gene III is prepared as follows: M13 delta gene III helperphage does not encode gene III protein, hence the phage(mid) displayingantibody fragments have a greater avidity of binding to antigen.Infectious M13 delta gene III particles are made by growing the helperphage in cells harboring a pUC19 derivative supplying the wild type geneIII protein during phage morphogenesis. The culture is incubated for 1hour at 37° C. without shaking and then for a further hour at 37° C.with shaking. Cells are spun down (IEC-Centra 8,400 r.p.m. for 10 min),resuspended in 300 ml 2xTY broth containing 100 μg ampicillin/ml and 25μg kanamycin/ml (2xTY-AMP-KAN) and grown overnight, shaking at 37° C.Phage particles are purified and concentrated from the culture medium bytwo PEG-precipitations (Sambrook et al., 1990), resuspended in 2 ml PBSand passed through a 0.45 μm filter (Minisart NML; Sartorius) to give afinal concentration of approximately 1013 transducing units/ml(ampicillin-resistant clones).

Panning of the Library. Immunotubes (Nunc) are coated overnight in PBSwith 4 ml of either 100 μg/ml or 10 μg/ml of a polypeptide of thepresent invention. Tubes are blocked with 2% Marvel-PBS for 2 hours at37° C. and then washed 3 times in PBS. Approximately 1013 TU of phage isapplied to the tube and incubated for 30 minutes at room temperaturetumbling on an over and under turntable and then left to stand foranother 1.5 hours. Tubes are washed 10 times with PBS 0.1% Tween-20 and10 times with PBS. Phage are eluted by adding 1 ml of 100 mMtriethylamine and rotating 15 minutes on an under and over turntableafter which the solution is immediately neutralized with 0.5 ml of 1.0MTris-HCl, pH 7.4. Phage are then used to infect 10 ml of mid-log E. coliTG1 by incubating eluted phage with bacteria for 30 minutes at 37° C.The E. coli are then plated on TYE plates containing 1% glucose and 100μg/ml ampicillin. The resulting bacterial library is then rescued withdelta gene 3 helper phage as described above to prepare phage for asubsequent round of selection. This process is then repeated for a totalof 4 rounds of affinity purification with tube-washing increased to 20times with PBS, 0.1% Tween-20 and 20 times with PBS for rounds 3 and 4.

Characterization of Binders. Eluted phage from the 3rd and 4th rounds ofselection are used to infect E. coli HB 2151 and soluble scFv isproduced (Marks, et al., 1991) from single colonies for assay. ELISAsare performed with microtitre plates coated with either 10 pg/ml of thepolypeptide of the present invention in 50 mM bicarbonate pH 9.6. Clonespositive in ELISA are further characterized by PCR fingerprinting (see,e.g., PCT publication WO 92/01047) and then by sequencing. These ELISApositive clones may also be further characterized by techniques known inthe art, such as, for example, epitope mapping, binding affinity,receptor signal transduction, ability to block or competitively inhibitantibody/antigen binding, and competitive agonistic or antagonisticactivity.

Example 33 Assays Detecting Stimulation or Inhibition of B CellProliferation and Differentiation

Generation of functional humoral immune responses requires both solubleand cognate signaling between B-lineage cells and theirmicroenvironment. Signals may impart a positive stimulus that allows aB-lineage cell to continue its programmed development, or a negativestimulus that instructs the cell to arrest its current developmentalpathway. To date, numerous stimulatory and inhibitory signals have beenfound to influence B cell responsiveness including IL-2, IL-4, IL-5,IL-6, IL-7, IL10, IL-13, IL-14 and IL-15. Interestingly, these signalsare by themselves weak effectors but can, in combination with variousco-stimulatory proteins, induce activation, proliferation,differentiation, homing, tolerance and death among B cell populations.

One of the best studied classes of B-cell co-stimulatory proteins is theTNF-superfamily. Within this family CD40, CD27, and CD30 along withtheir respective ligands CD154, CD70, and CD153 have been found toregulate a variety of immune responses. Assays which allow for thedetection and/or observation of the proliferation and differentiation ofthese B-cell populations and their precursors are valuable tools indetermining the effects various proteins may have on these B-cellpopulations in terms of proliferation and differentiation. Listed beloware two assays designed to allow for the detection of thedifferentiation, proliferation, or inhibition of B-cell populations andtheir precursors.

In Vitro Assay—Purified polypeptides of the invention, or truncatedforms thereof, is assessed for its ability to induce activation,proliferation, differentiation or inhibition and/or death in B-cellpopulations and their precursors. The activity of the polypeptides ofthe invention on purified human tonsillar B cells, measuredqualitatively over the dose range from 0.1 to 10,000 ng/mL, is assessedin a standard B-lymphocyte co-stimulation assay in which purifiedtonsillar B cells are cultured in the presence of either formalin-fixedStaphylococcus aureus Cowan I (SAC) or immobilized anti-human IgMantibody as the priming agent. Second signals such as IL-2 and IL-15synergize with SAC and IgM crosslinking to elicit B cell proliferationas measured by tritiated-thymidine incorporation. Novel synergizingagents can be readily identified using this assay. The assay involvesisolating human tonsillar B cells by magnetic bead (MACS) depletion ofCD3-positive cells. The resulting cell population is greater than 95% Bcells as assessed by expression of CD45R(B220).

Various dilutions of each sample are placed into individual wells of a96-well plate to which are added 105 B-cells suspended in culture medium(RPMI 1640 containing 10% FBS, 5×10-5M 2ME, 100 U/ml penicillin, 10ug/ml streptomycin, and 10-5 dilution of SAC) in a total volume of 150ul. Proliferation or inhibition is quantitated by a 20 h pulse (1uCi/well) with 3H-thymidine (6.7 Ci/mM) beginning 72 h post factoraddition. The positive and negative controls are IL2 and mediumrespectively.

In Vivo Assay—BALB/c mice are injected (i.p.) twice per day with bufferonly, or 2 mg/Kg of a polypeptide of the invention, or truncated formsthereof. Mice receive this treatment for 4 consecutive days, at whichtime they are sacrificed and various tissues and serum collected foranalyses. Comparison of H&E sections from normal spleens and spleenstreated with polypeptides of the invention identify the results of theactivity of the polypeptides on spleen cells, such as the diffusion ofperi-arterial lymphatic sheaths, and/or significant increases in thenucleated cellularity of the red pulp regions, which may indicate theactivation of the differentiation and proliferation of B-cellpopulations. Immunohistochemical studies using a B cell marker,anti-CD45R(B220), are used to determine whether any physiologicalchanges to splenic cells, such as splenic disorganization, are due toincreased B-cell representation within loosely defined B-cell zones thatinfiltrate established T-cell regions.

Flow cytometric analyses of the spleens from mice treated withpolypeptide is used to indicate whether the polypeptide specificallyincreases the proportion of ThB+, CD45R(B220)dull B cells over thatwhich is observed in control mice.

Likewise, a predicted consequence of increased mature B-cellrepresentation in vivo is a relative increase in serum Ig titers.Accordingly, serum IgM and IgA levels are compared between buffer andpolypeptide-treated mice.

One skilled in the art could easily modify the exemplified studies totest the activity of polynucleotides of the invention (e.g., genetherapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 34 T Cell Proliferation Assay

A CD3-induced proliferation assay is performed on PBMCs and is measuredby the uptake of 3H-thymidine. The assay is performed as follows.Ninety-six well plates are coated with 100 (I/well of mAb to CD3 (HIT3a,Pharmingen) or isotype-matched control mAb (B33.1) overnight at 4degrees C. (1 (g/ml in 0.05M bicarbonate buffer, pH 9.5), then washedthree times with PBS. PBMC are isolated by F/H gradient centrifugationfrom human peripheral blood and added to quadruplicate wells(5×104/well) of mAb coated plates in RPMI containing 10% FCS and P/S inthe presence of varying concentrations of polypeptides of the invention(total volume 200 ul). Relevant protein buffer and medium alone arecontrols. After 48 hr. culture at 37 degrees C., plates are spun for 2min. at 1000 rpm and 100 (I of supernatant is removed and stored −20degrees C. for measurement of IL-2 (or other cytokines) if effect onproliferation is observed. Wells are supplemented with 100 ul of mediumcontaining 0.5 uCi of 3H-thymidine and cultured at 37 degrees C. for18-24 hr. Wells are harvested and incorporation of 3H-thymidine used asa measure of proliferation. Anti-CD3 alone is the positive control forproliferation. IL-2 (100 U/ml) is also used as a control which enhancesproliferation. Control antibody which does not induce proliferation of Tcells is used as the negative controls for the effects of polypeptidesof the invention.

One skilled in the art could easily modify the exemplified studies totest the activity of polynucleotides of the invention (e.g., genetherapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 35 Effect of Polypeptides of the Invention on the Expression ofMHC Class II, Costimulatory and Adhesion Molecules and CellDifferentiation of Monocytes and Monocyte-Derived Human Dendritic Cells

Dendritic cells are generated by the expansion of proliferatingprecursors found in the peripheral blood: adherent PBMC or elutriatedmonocytic fractions are cultured for 7-10 days with GM-CSF (50 ng/ml)and IL-4 (20 ng/ml). These dendritic cells have the characteristicphenotype of immature cells (expression of CD1, CD80, CD86, CD40 and MHCclass II antigens). Treatment with activating factors, such as TNF-(,causes a rapid change in surface phenotype (increased expression of MHCclass I and II, costimulatory and adhesion molecules, downregulation ofFC(RII, upregulation of CD83). These changes correlate with increasedantigen-presenting capacity and with functional maturation of thedendritic cells.

FACS analysis of surface antigens is performed as follows. Cells aretreated 1-3 days with increasing concentrations of polypeptides of theinvention or LPS (positive control), washed with PBS containing 1% BSAand 0.02 mM sodium azide, and then incubated with 1:20 dilution ofappropriate FITC- or PE-labeled monoclonal antibodies for 30 minutes at4 degrees C. After an additional wash, the labeled cells are analyzed byflow cytometry on a FACScan (Becton Dickinson).

Effect on the production of cytokines. Cytokines generated by dendriticcells, in particular IL-12, are important in the initiation of T-celldependent immune responses. IL-12 strongly influences the development ofTh1 helper T-cell immune response, and induces cytotoxic T and NK cellfunction. An ELISA is used to measure the IL-12 release as follows.Dendritic cells (106/ml) are treated with increasing concentrations ofpolypeptides of the invention for 24 hours. LPS (100 ng/ml) is added tothe cell culture as positive control. Supernatants from the cellcultures are then collected and analyzed for IL-12 content usingcommercial ELISA kit (e.g., R & D Systems (Minneapolis, Minn.)). Thestandard protocols provided with the kits are used.

Effect on the expression of MHC Class II, costimulatory and adhesionmolecules. Three major families of cell surface antigens can beidentified on monocytes: adhesion molecules, molecules involved inantigen presentation, and Fc receptor. Modulation of the expression ofMHC class II antigens and other costimulatory molecules, such as B7 andICAM-1, may result in changes in the antigen presenting capacity ofmonocytes and ability to induce T cell activation. Increase expressionof Fc receptors may correlate with improved monocyte cytotoxic activity,cytokine release and phagocytosis.

FACS analysis is used to examine the surface antigens as follows.Monocytes are treated 1-5 days with increasing concentrations ofpolypeptides of the invention or LPS (positive control), washed with PBScontaining 1% BSA and 0.02 mM sodium azide, and then incubated with 1:20dilution of appropriate FITC- or PE-labeled monoclonal antibodies for 30minutes at 4 degrees C. After an additional wash, the labeled cells areanalyzed by flow cytometry on a FACScan (Becton Dickinson).

Monocyte activation and/or increased survival. Assays for molecules thatactivate (or alternatively, inactivate) monocytes and/or increasemonocyte survival (or alternatively, decrease monocyte survival) areknown in the art and may routinely be applied to determine whether amolecule of the invention functions as an inhibitor or activator ofmonocytes. Polypeptides, agonists, or antagonists of the invention canbe screened using the three assays described below. For each of theseassays, Peripheral blood mononuclear cells (PBMC) are purified fromsingle donor leukopacks (American Red Cross, Baltimore, Md.) bycentrifugation through a Histopaque gradient (Sigma). Monocytes areisolated from PBMC by counterflow centrifugal elutriation.

Monocyte Survival Assay. Human peripheral blood monocytes progressivelylose viability when cultured in absence of serum or other stimuli. Theirdeath results from internally regulated process (apoptosis). Addition tothe culture of activating factors, such as TNF-alpha dramaticallyimproves cell survival and prevents DNA fragmentation. Propidium iodide(PI) staining is used to measure apoptosis as follows. Monocytes arecultured for 48 hours in polypropylene tubes in serum-free medium(positive control), in the presence of 100 ng/ml TNF-alpha (negativecontrol), and in the presence of varying concentrations of the compoundto be tested. Cells are suspended at a concentration of 2×106/ml in PBScontaining PI at a final concentration of 5 (g/ml, and then incubated atroom temperature for 5 minutes before FACScan analysis. PI uptake hasbeen demonstrated to correlate with DNA fragmentation in thisexperimental paradigm.

Effect on cytokine release. An important function ofmonocytes/macrophages is their regulatory activity on other cellularpopulations of the immune system through the release of cytokines afterstimulation. An ELISA to measure cytokine release is performed asfollows. Human monocytes are incubated at a density of 5×105 cells/mlwith increasing concentrations of the a polypeptide of the invention andunder the same conditions, but in the absence of the polypeptide. ForIL-12 production, the cells are primed overnight with IFN (100 U/ml) inpresence of a polypeptide of the invention. LPS (10 ng/ml) is thenadded. Conditioned media are collected after 24 h and kept frozen untiluse. Measurement of TNF-alpha, IL-10, MCP-1 and IL-8 is then performedusing a commercially available ELISA kit (e.g., R & D Systems(Minneapolis, Minn.)) and applying the standard protocols provided withthe kit.

Oxidative burst. Purified monocytes are plated in 96-w plate at 2-1×105cell/well. Increasing concentrations of polypeptides of the inventionare added to the wells in a total volume of 0.2 ml culture medium (RPMI1640+10% FCS, glutamine and antibiotics). After 3 days incubation, theplates are centrifuged and the medium is removed from the wells. To themacrophage monolayers, 0.2 ml per well of phenol red solution (140 mMNaCl, 10 mM potassium phosphate buffer pH 7.0, 5.5 mM dextrose, 0.56 mMphenol red and 19 U/ml of HRPO) is added, together with the stimulant(200 nM PMA). The plates are incubated at 37 (C for 2 hours and thereaction is stopped by adding 20 μl 1N NaOH per well. The absorbance isread at 610 nm. To calculate the amount of H2O2 produced by themacrophages, a standard curve of a H2O2 solution of known molarity isperformed for each experiment.

One skilled in the art could easily modify the exemplified studies totest the activity of polynucleotides of the invention (e.g., genetherapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 36 Biological Effects of Human Phosphatase Polypeptides of theInvention

Astrocyte and Neuronal Assays

Recombinant polypeptides of the invention, expressed in Escherichia coliand purified as described above, can be tested for activity in promotingthe survival, neurite outgrowth, or phenotypic differentiation ofcortical neuronal cells and for inducing the proliferation of glialfibrillary acidic protein immunopositive cells, astrocytes. Theselection of cortical cells for the bioassay is based on the prevalentexpression of FGF-1 and FGF-2 in cortical structures and on thepreviously reported enhancement of cortical neuronal survival resultingfrom FGF-2 treatment. A thymidine incorporation assay, for example, canbe used to elucidate a polypeptide of the invention's activity on thesecells.

Moreover, previous reports describing the biological effects of FGF-2(basic FGF) on cortical or hippocampal neurons in vitro havedemonstrated increases in both neuron survival and neurite outgrowth(Walicke et al., “Fibroblast growth factor promotes survival ofdissociated hippocampal neurons and enhances neurite extension.” Proc.Natl. Acad. Sci. USA 83:3012-3016. (1986), assay herein incorporated byreference in its entirety). However, reports from experiments done onPC-12 cells suggest that these two responses are not necessarilysynonymous and may depend on not only which FGF is being tested but alsoon which receptor(s) are expressed on the target cells. Using theprimary cortical neuronal culture paradigm, the ability of a polypeptideof the invention to induce neurite outgrowth can be compared to theresponse achieved with FGF-2 using, for example, a thymidineincorporation assay.

Fibroblast and Endothelial Cell Assays.

Human lung fibroblasts are obtained from Clonetics (San Diego, Calif.)and maintained in growth media from Clonetics. Dermal microvascularendothelial cells are obtained from Cell Applications (San Diego,Calif.). For proliferation assays, the human lung fibroblasts and dermalmicrovascular endothelial cells can be cultured at 5,000 cells/well in a96-well plate for one day in growth medium. The cells are then incubatedfor one day in 0.1% BSA basal medium. After replacing the medium withfresh 0.1% BSA medium, the cells are incubated with the test proteinsfor 3 days. Alamar Blue (Alamar Biosciences, Sacramento, Calif.) isadded to each well to a final concentration of 10%. The cells areincubated for 4 hr. Cell viability is measured by reading in a CytoFluorfluorescence reader. For the PGE2 assays, the human lung fibroblasts arecultured at 5,000 cells/well in a 96-well plate for one day. After amedium change to 0.1% BSA basal medium, the cells are incubated withFGF-2 or polypeptides of the invention with or without IL-1 (for 24hours. The supernatants are collected and assayed for PGE2 by EIA kit(Cayman, Ann Arbor, Mich.). For the IL-6 assays, the human lungfibroblasts are cultured at 5,000 cells/well in a 96-well plate for oneday. After a medium change to 0.1% BSA basal medium, the cells areincubated with FGF-2 or with or without polypeptides of the inventionIL-1 (for 24 hours. The supernatants are collected and assayed for IL-6by ELISA kit (Endogen, Cambridge, Mass.).

Human lung fibroblasts are cultured with FGF-2 or polypeptides of theinvention for 3 days in basal medium before the addition of Alamar Blueto assess effects on growth of the fibroblasts. FGF-2 should show astimulation at 10-2500 ng/ml which can be used to compare stimulationwith polypeptides of the invention.

Parkinson Models.

The loss of motor function in Parkinson's disease is attributed to adeficiency of striatal dopamine resulting from the degeneration of thenigrostriatal dopaminergic projection neurons. An animal model forParkinson's that has been extensively characterized involves thesystemic administration of 1-methyl-4 phenyl 1,2,3,6-tetrahydropyridine(MPTP). In the CNS, MPTP is taken-up by astrocytes and catabolized bymonoamine oxidase B to 1-methyl-4-phenyl pyridine (MPP+) and released.Subsequently, MPP+ is actively accumulated in dopaminergic neurons bythe high-affinity reuptake transporter for dopamine. MPP+ is thenconcentrated in mitochondria by the electrochemical gradient andselectively inhibits nicotidamide adenine disphosphate: ubiquinoneoxidoreductionase (complex I), thereby interfering with electrontransport and eventually generating oxygen radicals.

It has been demonstrated in tissue culture paradigms that FGF-2 (basicFGF) has trophic activity towards nigral dopaminergic neurons (Ferrariet al., Dev. Biol. 1989). Recently, Dr. Unsicker's group hasdemonstrated that administering FGF-2 in gel foam implants in thestriatum results in the near complete protection of nigral dopaminergicneurons from the toxicity associated with MPTP exposure (Otto andUnsicker, J. Neuroscience, 1990).

Based on the data with FGF-2, polypeptides of the invention can beevaluated to determine whether it has an action similar to that of FGF-2in enhancing dopaminergic neuronal survival in vitro and it can also betested in vivo for protection of dopaminergic neurons in the striatumfrom the damage associated with MPTP treatment. The potential effect ofa polypeptide of the invention is first examined in vitro in adopaminergic neuronal cell culture paradigm. The cultures are preparedby dissecting the midbrain floor plate from gestation day 14 Wistar ratembryos. The tissue is dissociated with trypsin and seeded at a densityof 200,000 cells/cm2 on polyorthinine-laminin coated glass coverslips.The cells are maintained in Dulbecco's Modified Eagle's medium and F12medium containing hormonal supplements (N1). The cultures are fixed withparaformaldehyde after 8 days in vitro and are processed for tyrosinehydroxylase, a specific marker for dopaminergic neurons,immunohistochemical staining. Dissociated cell cultures are preparedfrom embryonic rats. The culture medium is changed every third day andthe factors are also added at that time.

Since the dopaminergic neurons are isolated from animals at gestationday 14, a developmental time which is past the stage when thedopaminergic precursor cells are proliferating, an increase in thenumber of tyrosine hydroxylase immunopositive neurons would represent anincrease in the number of dopaminergic neurons surviving in vitro.Therefore, if a polypeptide of the invention acts to prolong thesurvival of dopaminergic neurons, it would suggest that the polypeptidemay be involved in Parkinson's Disease.

One skilled in the art could easily modify the exemplified studies totest the activity of polynucleotides of the invention (e.g., genetherapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 37 The Effect of the human Phosphatase Polypeptides of theInvention on the Growth of Vascular Endothelial Cells

On day 1, human umbilical vein endothelial cells (HUVEC) are seeded at2-5×104 cells/35 mm dish density in M199 medium containing 4% fetalbovine serum (FBS), 16 units/ml heparin, and 50 units/ml eridothelialcell growth supplements (ECGS, Biotechnique, Inc.). On day 2, the mediumis replaced with M199 containing 10% FBS, 8 units/ml heparin. Apolypeptide having the amino acid sequence of SEQ ID NO:Y, and positivecontrols, such as VEGF and basic FGF (bFGF) are added, at varyingconcentrations. On days 4 and 6, the medium is replaced. On day 8, cellnumber is determined with a Coulter Counter.

An increase in the number of HUVEC cells indicates that the polypeptideof the invention may proliferate vascular endothelial cells.

One skilled in the art could easily modify the exemplified studies totest the activity of polynucleotides of the invention (e.g., genetherapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 38 Stimulatory Effect of Polypeptides of the Invention on theProliferation of Vascular Endothelial Cells

For evaluation of mitogenic activity of growth factors, the colorimetricMTS(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)assay with the electron coupling reagent PMS (phenazine methosulfate)was performed (CellTiter 96 AQ, Promega). Cells are seeded in a 96-wellplate (5,000 cells/well) in 0.1 mL serum-supplemented medium and areallowed to attach overnight. After serum-starvation for 12 hours in 0.5%FBS, conditions (bFGF, VEGF165 or a polypeptide of the invention in 0.5%FBS) with or without Heparin (8 U/ml) are added to wells for 48 hours.20 mg of MTS/PMS mixture (1:0.05) are added per well and allowed toincubate for 1 hour at 37° C. before measuring the absorbance at 490 nmin an ELISA plate reader. Background absorbance from control wells (somemedia, no cells) is subtracted, and seven wells are performed inparallel for each condition. See, Leak et al. In Vitro Cell. Dev. Biol.30A:512-518 (1994).

One skilled in the art could easily modify the exemplified studies totest the activity of polynucleotides of the invention (e.g., genetherapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 39 Inhibition of PDGF-Induced Vascular Smooth Muscle CellProliferation Stimulatory Effect

HAoSMC proliferation can be measured, for example, by BrdUrdincorporation. Briefly, subconfluent, quiescent cells grown on the4-chamber slides are transfected with CRP or FITC-labeled AT2-3LP. Then,the cells are pulsed with 10% calf serum and 6 mg/ml BrdUrd. After 24 h,immunocytochemistry is performed by using BrdUrd Staining Kit (ZymedLaboratories). In brief, the cells are incubated with the biotinylatedmouse anti-BrdUrd antibody at 4 degrees C. for 2 h after being exposedto denaturing solution and then incubated with thestreptavidin-peroxidase and diaminobenzidine. After counterstaining withhematoxylin, the cells are mounted for microscopic examination, and theBrdUrd-positive cells are counted. The BrdUrd index is calculated as apercent of the BrdUrd-positive cells to the total cell number. Inaddition, the simultaneous detection of the BrdUrd staining (nucleus)and the FITC uptake (cytoplasm) is performed for individual cells by theconcomitant use of bright field illumination and dark field-UVfluorescent illumination. See, Hayashida et al., J. Biol. Chem. 6:271(36):21985-21992 (1996).

One skilled in the art could easily modify the exemplified studies totest the activity of polynucleotides of the invention (e.g., genetherapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 40 Stimulation of Endothelial Migration

This example will be used to explore the possibility that a polypeptideof the invention may stimulate lymphatic endothelial cell migration.

Endothelial cell migration assays are performed using a 48 wellmicrochemotaxis chamber (Neuroprobe Inc., Cabin John, M D; Falk, W., etal., J. Immunological Methods 1980; 33:239-247).Polyvinylpyrrolidone-free polycarbonate filters with a pore size of 8 um(Nucleopore Corp. Cambridge, Mass.) are coated with 0.1% gelatin for atleast 6 hours at room temperature and dried under sterile air. Testsubstances are diluted to appropriate concentrations in M199supplemented with 0.25% bovine serum albumin (BSA), and 25 ul of thefinal dilution is placed in the lower chamber of the modified Boydenapparatus. Subconfluent, early passage (2-6) HUVEC or BMEC cultures arewashed and trypsinized for the minimum time required to achieve celldetachment. After placing the filter between lower and upper chamber,2.5×105 cells suspended in 50 ul M199 containing 1% FBS are seeded inthe upper compartment. The apparatus is then incubated for 5 hours at37° C. in a humidified chamber with 5% CO2 to allow cell migration.After the incubation period, the filter is removed and the upper side ofthe filter with the non-migrated cells is scraped with a rubberpoliceman. The filters are fixed with methanol and stained with a Giemsasolution (Diff-Quick, Baxter, McGraw Park, Ill.). Migration isquantified by counting cells of three random high-power fields (40×) ineach well, and all groups are performed in quadruplicate.

One skilled in the art could easily modify the exemplified studies totest the activity of polynucleotides of the invention (e.g., genetherapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 41 Stimulation of Nitric Oxide Production by Endothelial Cells

Nitric oxide released by the vascular endothelium is believed to be amediator of vascular endothelium relaxation. Thus, activity of apolypeptide of the invention can be assayed by determining nitric oxideproduction by endothelial cells in response to the polypeptide.

Nitric oxide is measured in 96-well plates of confluent microvascularendothelial cells after 24 hours starvation and a subsequent 4 hrexposure to various levels of a positive control (such as VEGF-1) andthe polypeptide of the invention. Nitric oxide in the medium isdetermined by use of the Griess reagent to measure total nitrite afterreduction of nitric oxide-derived nitrate by nitrate reductase. Theeffect of the polypeptide of the invention on nitric oxide release isexamined on HUVEC.

Briefly, NO release from cultured HUVEC monolayer is measured with aNO-specific polarographic electrode connected to a NO meter (Iso-NO,World Precision Instruments Inc.) (1049). Calibration of the NO elementsis performed according to the following equation:2KNO2+2KI+2H2SO4 6 2NO+I2+2H2O+2K2SO4

The standard calibration curve is obtained by adding gradedconcentrations of KNO2 (0, 5, 10, 25, 50, 100, 250, and 500 nmol/L) intothe calibration solution containing K1 and H2SO4. The specificity of theIso-NO electrode to NO is previously determined by measurement of NOfrom authentic NO gas (1050). The culture medium is removed and HUVECsare washed twice with Dulbecco's phosphate buffered saline. The cellsare then bathed in 5 ml of filtered Krebs-Henseleit solution in 6-wellplates, and the cell plates are kept on a slide warmer (Lab LineInstruments Inc.) To maintain the temperature at 37° C. The NO sensorprobe is inserted vertically into the wells, keeping the tip of theelectrode 2 mm under the surface of the solution, before addition of thedifferent conditions. S-nitroso acetyl penicillamin (SNAP) is used as apositive control. The amount of released NO is expressed as picomolesper 1×106 endothelial cells. All values reported are means of four tosix measurements in each group (number of cell culture wells). See, Leaket al. Biochem. and Biophys. Res. Comm. 217:96-105 (1995).

One skilled in the art could easily modify the exemplified studies totest the activity of polynucleotides of the invention (e.g., genetherapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 42 Effect of Human Phosphatase Polypepides of the Invention onCord Formation in Angiogenesis

Another step in angiogenesis is cord formation, marked bydifferentiation of endothelial cells. This bioassay measures the abilityof microvascular endothelial cells to form capillary-like structures(hollow structures) when cultured in vitro.

CADMEC (microvascular endothelial cells) are purchased from CellApplications, Inc. as proliferating (passage 2) cells and are culturedin Cell Applications' CADMEC Growth Medium and used at passage 5. Forthe in vitro angiogenesis assay, the wells of a 48-well cell cultureplate are coated with Cell Applications' Attachment Factor Medium (200ml/well) for 30 min. at 37° C. CADMEC are seeded onto the coated wellsat 7,500 cells/well and cultured overnight in Growth Medium. The GrowthMedium is then replaced with 300 mg Cell Applications' Chord FormationMedium containing control buffer or a polypeptide of the invention (0.1to 100 ng/ml) and the cells are cultured for an additional 48 hr. Thenumbers and lengths of the capillary-like chords are quantitated throughuse of the Boeckeler VIA-170 video image analyzer. All assays are donein triplicate.

Commercial (R&D) VEGF (50 ng/ml) is used as a positive control.b-esteradiol (1 ng/ml) is used as a negative control. The appropriatebuffer (without protein) is also utilized as a control.

One skilled in the art could easily modify the exemplified studies totest the activity of polynucleotides of the invention (e.g., genetherapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 43 Angiogenic Effect on Chick Chorioallantoic Membrane

Chick chorioallantoic membrane (CAM) is a well-established system toexamine angiogenesis. Blood vessel formation on CAM is easily visibleand quantifiable. The ability of polypeptides of the invention tostimulate angiogenesis in CAM can be examined.

Fertilized eggs of the White Leghorn chick (Gallus gallus) and theJapanese qual (Coturnix coturnix) are incubated at 37.8° C. and 80%humidity. Differentiated CAM of 16-day-old chick and 13-day-old qualembryos is studied with the following methods.

On Day 4 of development, a window is made into the egg shell of chickeggs. The embryos are checked for normal development and the eggs sealedwith cellotape. They are further incubated until Day 13. Thermanoxcoverslips (Nunc, Naperville, Ill.) are cut into disks of about 5 mm indiameter. Sterile and salt-free growth factors are dissolved indistilled water and about 3.3 mg/5 ml are pipetted on the disks. Afterair-drying, the inverted disks are applied on CAM. After 3 days, thespecimens are fixed in 3% glutaraldehyde and 2% formaldehyde and rinsedin 0.12 M sodium cacodylate buffer. They are photographed with a stereomicroscope [Wild M8] and embedded for semi- and ultrathin sectioning asdescribed above. Controls are performed with carrier disks alone.

One skilled in the art could easily modify the exemplified studies totest the activity of polynucleotides of the invention (e.g., genetherapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 44 Angiogenesis Assay Using a Matrigel Implant in Mouse

In vivo angiogenesis assay of a polypeptide of the invention measuresthe ability of an existing capillary network to form new vessels in animplanted capsule of murine extracellular matrix material (Matrigel).The protein is mixed with the liquid Matrigel at 4 degree C. and themixture is then injected subcutaneously in mice where it solidifies.After 7 days, the solid “plug” of Matrigel is removed and examined forthe presence of new blood vessels. Matrigel is purchased from BectonDickinson Labware/Collaborative Biomedical Products.

When thawed at 4 degree C. the Matrigel material is a liquid. TheMatrigel is mixed with a polypeptide of the invention at 150 ng/ml at 4degrees C. and drawn into cold 3 ml syringes. Female C57Bl/6 miceapproximately 8 weeks old are injected with the mixture of Matrigel andexperimental protein at 2 sites at the midventral aspect of the abdomen(0.5 ml/site). After 7 days, the mice are sacrificed by cervicaldislocation, the Matrigel plugs are removed and cleaned (i.e., allclinging membranes and fibrous tissue is removed). Replicate whole plugsare fixed in neutral buffered 10% formaldehyde, embedded in paraffin andused to produce sections for histological examination after stainingwith Masson's Trichrome. Cross sections from 3 different regions of eachplug are processed. Selected sections are stained for the presence ofvWF. The positive control for this assay is bovine basic FGF (150ng/ml). Matrigel alone is used to determine basal levels ofangiogenesis.

One skilled in the art could easily modify the exemplified studies totest the activity of polynucleotides of the invention (e.g., genetherapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 45 Rescue of Ischemia in Rabbit Lower Limb Model

To study the in vivo effects of polynucleotides and polypeptides of theinvention on ischemia, a rabbit hindlimb ischemia model is created bysurgical removal of one femoral arteries as described previously(Takeshita et al., Am J. Pathol 147:1649-1660 (1995)). The excision ofthe femoral artery results in retrograde propagation of thrombus andocclusion of the external iliac artery. Consequently, blood flow to theischemic limb is dependent upon collateral vessels originating from theinternal iliac artery (Takeshita et al. Am J. Pathol 147:1649-1660(1995)). An interval of 10 days is allowed for post-operative recoveryof rabbits and development of endogenous collateral vessels. At 10 daypost-operatively (day 0), after performing a baseline angiogram, theinternal iliac artery of the ischemic limb is transfected with 500 mgnaked expression plasmid containing a polynucleotide of the invention byarterial gene transfer technology using a hydrogel-coated ballooncatheter as described (Riessen et al. Hum Gene Ther. 4:749-758 (1993);Leclerc et al. J. Clin. Invest. 90: 936-944 (1992)). When a polypeptideof the invention is used in the treatment, a single bolus of 500 mgpolypeptide of the invention or control is delivered into the internaliliac artery of the ischemic limb over a period of 1 min. through aninfusion catheter. On day 30, various parameters are measured in theserabbits: (a) BP ratio—The blood pressure ratio of systolic pressure ofthe ischemic limb to that of normal limb; (b) Blood Flow and FlowReserve—Resting FL: the blood flow during undilated condition and MaxFL: the blood flow during fully dilated condition (also an indirectmeasure of the blood vessel amount) and Flow Reserve is reflected by theratio of max FL: resting FL; (c) Angiographic Score—This is measured bythe angiogram of collateral vessels. A score is determined by thepercentage of circles in an overlaying grid that with crossing opacifiedarteries divided by the total number m the rabbit thigh; (d) Capillarydensity—The number of collateral capillaries determined in lightmicroscopic sections taken from hindlimbs.

One skilled in the art could easily modify the exemplified studies totest the activity of polynucleotides of the invention (e.g., genetherapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 46 Effect of Polypeptides of the Invention on Vasodilation

Since dilation of vascular endothelium is important in reducing bloodpressure, the ability of polypeptides of the invention to affect theblood pressure in spontaneously hypertensive rats (SHR) is examined.Increasing doses (0, 10, 30, 100, 300, and 900 mg/kg) of thepolypeptides of the invention are administered to 13-14 week oldspontaneously hypertensive rats (SHR). Data are expressed as themean+/−SEM. Statistical analysis are performed with a paired t-test andstatistical significance is defined as p<0.05 vs. the response to bufferalone.

One skilled in the art could easily modify the exemplified studies totest the activity of polynucleotides of the invention (e.g., genetherapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 47 Rat Ischemic Skin Flap Model

The evaluation parameters include skin blood flow, skin temperature, andfactor VIII immunohistochemistry or endothelial alkaline phosphatasereaction. Expression of polypeptides of the invention, during the skinischemia, is studied using in situ hybridization. The study in thismodel is divided into three parts as follows:

-   -   a) Ischemic skin    -   b) Ischemic skin wounds    -   c) Normal wounds        The experimental protocol includes:    -   a) Raising a 3×4 cm, single pedicle full-thickness random skin        flap (myocutaneous flap over the lower back of the animal).    -   b) An excisional wounding (4-6 mm in diameter) in the ischemic        skin (skin-flap).    -   c) Topical treatment with a polypeptide of the invention of the        excisional wounds (day 0, 1, 2, 3, 4 post-wounding) at the        following various dosage ranges: 1 mg to 100 mg.    -   d) Harvesting the wound tissues at day 3, 5, 7, 10, 14 and 21        post-wounding for histological, immunohistochemical, and in situ        studies.

One skilled in the art could easily modify the exemplified studies totest the activity of polynucleotides of the invention (e.g., genetherapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 48 Peripheral Arterial Disease Model

Angiogenic therapy using a polypeptide of the invention is a noveltherapeutic strategy to obtain restoration of blood flow around theischemia in case of peripheral arterial diseases. The experimentalprotocol includes:

-   -   a) One side of the femoral artery is ligated to create ischemic        muscle of the hindlimb, the other side of hindlimb serves as a        control.    -   b) a polypeptide of the invention, in a dosage range of 20        mg-500 mg, is delivered intravenously and/or intramuscularly 3        times (perhaps more) per week for 2-3 weeks.    -   c) The ischemic muscle tissue is collected after ligation of the        femoral artery at 1, 2, and 3 weeks for the analysis of        expression of a polypeptide of the invention and histology.        Biopsy is also performed on the other side of normal muscle of        the contralateral hindlimb.

One skilled in the art could easily modify the exemplified studies totest the activity of polynucleotides of the invention (e.g., genetherapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 49 Ischemic Myocardial Disease Model

A polypeptide of the invention is evaluated as a potent mitogen capableof stimulating the development of collateral vessels, and restructuringnew vessels after coronary artery occlusion. Alteration of expression ofthe polypeptide is investigated in situ. The experimental protocolincludes:

-   -   a) The heart is exposed through a left-side thoracotomy in the        rat. Immediately, the left coronary artery is occluded with a        thin suture (6-0) and the thorax is closed.    -   b) a polypeptide of the invention, in a dosage range of 20        mg-500 mg, is delivered intravenously and/or intramuscularly 3        times (perhaps more) per week for 2-4 weeks.    -   c) Thirty days after the surgery, the heart is removed and        cross-sectioned for morphometric and in situ analyzes.

One skilled in the art could easily modify the exemplified studies totest the activity of polynucleotides of the invention (e.g., genetherapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 50 Rat Corneal Wound Healing Model

This animal model shows the effect of a polypeptide of the invention onneovascularization. The experimental protocol includes:

-   -   a) Making a 1-1.5 mm long incision from the center of cornea        into the stromal layer.    -   b) Inserting a spatula below the lip of the incision facing the        outer corner of the eye.    -   c) Making a pocket (its base is 1-1.5 mm form the edge of the        eye).    -   d) Positioning a pellet, containing 50 ng-5 ug of a polypeptide        of the invention, within the pocket.    -   e) Treatment with a polypeptide of the invention can also be        applied topically to the corneal wounds in a dosage range of 20        mg-500 mg (daily treatment for five days).

One skilled in the art could easily modify the exemplified studies totest the activity of polynucleotides of the invention (e.g., genetherapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 51 Diabetic Mouse and Glucocorticoid-Impaired Wound HealingModels

A. Diabetic db+/db+ Mouse Model.

To demonstrate that a polypeptide of the invention accelerates thehealing process, the genetically diabetic mouse model of wound healingis used. The full thickness wound healing model in the db+/db+ mouse isa well characterized, clinically relevant and reproducible model ofimpaired wound healing. Healing of the diabetic wound is dependent onformation of granulation tissue and re-epithelialization rather thancontraction (Gartner, M. H. et al., J. Surg. Res. 52:389 (1992);Greenhalgh, D. G. et al., Am. J. Pathol. 136:1235 (1990)).

The diabetic animals have many of the characteristic features observedin Type II diabetes mellitus. Homozygous (db+/db+) mice are obese incomparison to their normal heterozygous (db+/+m) littermates. Mutantdiabetic (db+/db+) mice have a single autosomal recessive mutation onchromosome 4 (db+) (Coleman et al. Proc. Natl. Acad. Sci. USA 77:283-293(1982)). Animals show polyphagia, polydipsia and polyuria. Mutantdiabetic mice (db+/db+) have elevated blood glucose, increased or normalinsulin levels, and suppressed cell-mediated immunity (Mandel et al., J.Immunol. 120:1375 (1978); Debray-Sachs, M. et al., Clin. Exp. Immunol.51(1):1-7 (1983); Leiter et al., Am. J. of Pathol. 114:46-55 (1985)).Peripheral neuropathy, myocardial complications, and microvascularlesions, basement membrane thickening and glomerular filtrationabnormalities have been described in these animals (Norido, F. et al.,Exp. Neurol. 83(2):221-232 (1984); Robertson et al., Diabetes29(1):60-67 (1980); Giacomelli et al., Lab Invest. 40(4):460-473 (1979);Coleman, D. L. Diabetes 31 (Suppl):1-6 (1982)). These homozygousdiabetic mice develop hyperglycemia that is resistant to insulinanalogous to human type II diabetes (Mandel et al., J. Immunol.120:1375-1377 (1978)).

The characteristics observed in these animals suggests that healing inthis model may be similar to the healing observed in human diabetes(Greenhalgh, et al., Am. J. of Pathol. 136:1235-1246 (1990)).

Genetically diabetic female C57BL/KsJ (db+/db+) mice and theirnon-diabetic (db+/+m) heterozygous littermates are used in this study(Jackson Laboratories). The animals are purchased at 6 weeks of age andare 8 weeks old at the beginning of the study. Animals are individuallyhoused and received food and water ad libitum. All manipulations areperformed using aseptic techniques. The experiments are conductedaccording to the rules and guidelines of Bristol-Myers Squibb Company'sInstitutional Animal Care and Use Committee and the Guidelines for theCare and Use of Laboratory Animals.

Wounding protocol is performed according to previously reported methods(Tsuboi, R. and Rifkin, D. B., J. Exp. Med. 172:245-251 (1990)).Briefly, on the day of wounding, animals are anesthetized with anintraperitoneal injection of Avertin (0.01 mg/mL), 2,2,2-tribromoethanoland 2-methyl-2-butanol dissolved in deionized water. The dorsal regionof the animal is shaved and the skin washed with 70% ethanol solutionand iodine. The surgical area is dried with sterile gauze prior towounding. An 8 mm full-thickness wound is then created using a Keyestissue punch. Immediately following wounding, the surrounding skin isgently stretched to eliminate wound expansion. The wounds are left openfor the duration of the experiment. Application of the treatment isgiven topically for 5 consecutive days commencing on the day ofwounding. Prior to treatment, wounds are gently cleansed with sterilesaline and gauze sponges.

Wounds are visually examined and photographed at a fixed distance at theday of surgery and at two day intervals thereafter. Wound closure isdetermined by daily measurement on days 1-5 and on day 8. Wounds aremeasured horizontally and vertically using a calibrated Jameson caliper.Wounds are considered healed if granulation tissue is no longer visibleand the wound is covered by a continuous epithelium.

A polypeptide of the invention is administered using at a rangedifferent doses, from 4 mg to 500 mg per wound per day for 8 days invehicle. Vehicle control groups received 50 mL of vehicle solution.

Animals are euthanized on day 8 with an intraperitoneal injection ofsodium pentobarbital (300 mg/kg). The wounds and surrounding skin arethen harvested for histology and immunohistochemistry. Tissue specimensare placed in 10% neutral buffered formalin in tissue cassettes betweenbiopsy sponges for further processing.

Three groups of 10 animals each (5 diabetic and 5 non-diabetic controls)are evaluated: 1) Vehicle placebo control, 2) untreated group, and 3)treated group.

Wound closure is analyzed by measuring the area in the vertical andhorizontal axis and obtaining the total square area of the wound.Contraction is then estimated by establishing the differences betweenthe initial wound area (day 0) and that of post treatment (day 8). Thewound area on day 1 is 64 mm2, the corresponding size of the dermalpunch. Calculations are made using the following formula:[Open area on day 8]−[Open area on day 1]/[Open area on day 1]

Specimens are fixed in 10% buffered formalin and paraffin embeddedblocks are sectioned perpendicular to the wound surface (5 mm) and cutusing a Reichert-Jung microtome. Routine hematoxylin-eosin (H&E)staining is performed on cross-sections of bisected wounds. Histologicexamination of the wounds are used to assess whether the healing processand the morphologic appearance of the repaired skin is altered bytreatment with a polypeptide of the invention. This assessment includedverification of the presence of cell accumulation, inflammatory cells,capillaries, fibroblasts, re-epithelialization and epidermal maturity(Greenhalgh, D. G. et al., Am. J. Pathol. 136:1235 (1990)). A calibratedlens micrometer is used by a blinded observer.

Tissue sections are also stained immunohistochemically with a polyclonalrabbit anti-human keratin antibody using ABC Elite detection system.Human skin is used as a positive tissue control while non-immune IgG isused as a negative control. Keratinocyte growth is determined byevaluating the extent of reepithelialization of the wound using acalibrated lens micrometer.

Proliferating cell nuclear antigen/cyclin (PCNA) in skin specimens isdemonstrated by using anti-PCNA antibody (1:50) with an ABC Elitedetection system. Human colon cancer can serve as a positive tissuecontrol and human brain tissue can be used as a negative tissue control.Each specimen includes a section with omission of the primary antibodyand substitution with non-immune mouse IgG. Ranking of these sections isbased on the extent of proliferation on a scale of 0-8, the lower sideof the scale reflecting slight proliferation to the higher sidereflecting intense proliferation.

Experimental data are analyzed using an unpaired t test. A p value of<0.05 is considered significant.

B. Steroid Impaired Rat Model

The inhibition of wound healing by steroids has been well documented invarious in vitro and in vivo systems (Wahl, Glucocorticoids and Woundhealing. In: Anti-Inflammatory Steroid Action: Basic and ClinicalAspects. 280-302 (1989); Wahl et al., J. Immunol. 115: 476-481 (1975);Werb et al., J. Exp. Med. 147:1684-1694 (1978)). Glucocorticoids retardwound healing by inhibiting angiogenesis, decreasing vascularpermeability (Ebert et al., An. Intern. Med. 37:701-705 (1952)),fibroblast proliferation, and collagen synthesis (Beck et al., GrowthFactors. 5: 295-304 (1991); Haynes et al., J. Clin. Invest. 61: 703-797(1978)) and producing a transient reduction of circulating monocytes(Haynes et al., J. Clin. Invest. 61: 703-797 (1978); Wahl,“Glucocorticoids and wound healing”, In: Antiinflammatory SteroidAction: Basic and Clinical Aspects, Academic Press, New York, pp.280-302 (1989)). The systemic administration of steroids to impairedwound healing is a well establish phenomenon in rats (Beck et al.,Growth Factors. 5: 295-304 (1991); Haynes et al., J. Clin. Invest. 61:703-797 (1978); Wahl, “Glucocorticoids and wound healing”, In:Antiinflammatory Steroid Action: Basic and Clinical Aspects, AcademicPress, New York, pp. 280-302 (1989); Pierce et al., Proc. Natl. Acad.Sci. USA 86: 2229-2233 (1989)).

To demonstrate that a polypeptide of the invention can accelerate thehealing process, the effects of multiple topical applications of thepolypeptide on full thickness excisional skin wounds in rats in whichhealing has been impaired by the systemic administration ofmethylprednisolone is assessed.

Young adult male Sprague Dawley rats weighing 250-300 g (Charles RiverLaboratories) are used in this example. The animals are purchased at 8weeks of age and are 9 weeks old at the beginning of the study. Thehealing response of rats is impaired by the systemic administration ofmethylprednisolone (17 mg/kg/rat intramuscularly) at the time ofwounding. Animals are individually housed and received food and water adlibitum. All manipulations are performed using aseptic techniques. Thisstudy would be conducted according to the rules and guidelines ofBristol-Myers Squibb Corporations Guidelines for the Care and Use ofLaboratory Animals.

The wounding protocol is followed according to section A, above. On theday of wounding, animals are anesthetized with an intramuscularinjection of ketamine (50 mg/kg) and xylazine (5 mg/kg). The dorsalregion of the animal is shaved and the skin washed with 70% ethanol andiodine solutions. The surgical area is dried with sterile gauze prior towounding. An 8 mm full-thickness wound is created using a Keyes tissuepunch. The wounds are left open for the duration of the experiment.Applications of the testing materials are given topically once a day for7 consecutive days commencing on the day of wounding and subsequent tomethylprednisolone administration. Prior to treatment, wounds are gentlycleansed with sterile saline and gauze sponges.

Wounds are visually examined and photographed at a fixed distance at theday of wounding and at the end of treatment. Wound closure is determinedby daily measurement on days 1-5 and on day 8. Wounds are measuredhorizontally and vertically using a calibrated Jameson caliper. Woundsare considered healed if granulation tissue is no longer visible and thewound is covered by a continuous epithelium.

The polypeptide of the invention is administered using at a rangedifferent doses, from 4 mg to 500 mg per wound per day for 8 days invehicle. Vehicle control groups received 50 mL of vehicle solution.

Animals are euthanized on day 8 with an intraperitoneal injection ofsodium pentobarbital (300 mg/kg). The wounds and surrounding skin arethen harvested for histology. Tissue specimens are placed in 10% neutralbuffered formalin in tissue cassettes between biopsy sponges for furtherprocessing.

Four groups of 10 animals each (5 with methylprednisolone and 5 withoutglucocorticoid) are evaluated: 1) Untreated group 2) Vehicle placebocontrol 3) treated groups.

Wound closure is analyzed by measuring the area in the vertical andhorizontal axis and obtaining the total area of the wound. Closure isthen estimated by establishing the differences between the initial woundarea (day 0) and that of post treatment (day 8). The wound area on day 1is 64 mm2, the corresponding size of the dermal punch. Calculations aremade using the following formula:[Open area on day 8]−[Open area on day 1]/[Open area on day 1]

Specimens are fixed in 10% buffered formalin and paraffin embeddedblocks are sectioned perpendicular to the wound surface (5 mm) and cutusing an Olympus microtome. Routine hematoxylin-eosin (H&E) staining isperformed on cross-sections of bisected wounds. Histologic examinationof the wounds allows assessment of whether the healing process and themorphologic appearance of the repaired skin is improved by treatmentwith a polypeptide of the invention. A calibrated lens micrometer isused by a blinded observer to determine the distance of the wound gap.

Experimental data are analyzed using an unpaired t test. A p value of<0.05 is considered significant.

One skilled in the art could easily modify the exemplified studies totest the activity of polynucleotides of the invention (e.g., genetherapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 52 Lymphedema Animal Model

The purpose of this experimental approach is to create an appropriateand consistent lymphedema model for testing the therapeutic effects of apolypeptide of the invention in lymphangiogenesis and re-establishmentof the lymphatic circulatory system in the rat hind limb. Effectivenessis measured by swelling volume of the affected limb, quantification ofthe amount of lymphatic vasculature, total blood plasma protein, andhistopathology. Acute lymphedema is observed for 7-10 days. Perhaps moreimportantly, the chronic progress of the edema is followed for up to 3-4weeks.

Prior to beginning surgery, blood sample is drawn for proteinconcentration analysis. Male rats weighing approximately ˜350 g aredosed with Pentobarbital. Subsequently, the right legs are shaved fromknee to hip. The shaved area is swabbed with gauze soaked in 70% EtOH.Blood is drawn for serum total protein testing. Circumference andvolumetric measurements are made prior to injecting dye into paws aftermarking 2 measurement levels (0.5 cm above heel, at mid-pt of dorsalpaw). The intradermal dorsum of both right and left paws are injectedwith 0.05 ml of 1% Evan's Blue. Circumference and volumetricmeasurements are then made following injection of dye into paws.

Using the knee joint as a landmark, a mid-leg inguinal incision is madecircumferentially allowing the femoral vessels to be located. Forcepsand hemostats are used to dissect and separate the skin flaps. Afterlocating the femoral vessels, the lymphatic vessel that runs along sideand underneath the vessel(s) is located. The main lymphatic vessels inthis area are then electrically coagulated suture ligated.

Using a microscope, muscles in back of the leg (near the semitendinosisand adductors) are bluntly dissected. The popliteal lymph node is thenlocated. The 2 proximal and 2 distal lymphatic vessels and distal bloodsupply of the popliteal node are then and ligated by suturing. Thepopliteal lymph node, and any accompanying adipose tissue, is thenremoved by cutting connective tissues.

Care is taken to control any mild bleeding resulting from thisprocedure. After lymphatics are occluded, the skin flaps are sealed byusing liquid skin (Vetbond) (AJ Buck). The separated skin edges aresealed to the underlying muscle tissue while leaving a gap of ˜0.5 cmaround the leg. Skin also may be anchored by suturing to underlyingmuscle when necessary.

To avoid infection, animals are housed individually with mesh (nobedding). Recovering animals are checked daily through the optimaledematous peak, which typically occurred by day 5-7. The plateauedematous peak are then observed. To evaluate the intensity of thelymphedema, the circumference and volumes of 2 designated places on eachpaw before operation and daily for 7 days are measured. The effectplasma proteins on lymphedema is determined and whether protein analysisis a useful testing perimeter is also investigated. The weights of bothcontrol and edematous limbs are evaluated at 2 places. Analysis isperformed in a blind manner.

Circumference Measurements: Under brief gas anesthetic to prevent limbmovement, a cloth tape is used to measure limb circumference.Measurements are done at the ankle bone and dorsal paw by 2 differentpeople then those 2 readings are averaged. Readings are taken from bothcontrol and edematous limbs.

Volumetric Measurements: On the day of surgery, animals are anesthetizedwith Pentobarbital and are tested prior to surgery. For dailyvolumetrics animals are under brief halothane anesthetic (rapidimmobilization and quick recovery), both legs are shaved and equallymarked using waterproof marker on legs. Legs are first dipped in water,then dipped into instrument to each marked level then measured by Buxcoedema software (Chen/Victor). Data is recorded by one person, while theother is dipping the limb to marked area.

Blood-plasma protein measurements: Blood is drawn, spun, and serumseparated prior to surgery and then at conclusion for total protein andCa2+ comparison.

Limb Weight Comparison: After drawing blood, the animal is prepared fortissue collection. The limbs are amputated using a quillitine, then bothexperimental and control legs are cut at the ligature and weighed. Asecond weighing is done as the tibio-cacaneal joint is disarticulatedand the foot is weighed.

Histological Preparations: The transverse muscle located behind the knee(popliteal) area is dissected and arranged in a metal mold, filled withfreezeGel, dipped into cold methylbutane, placed into labeled samplebags at −80EC until sectioning. Upon sectioning, the muscle is observedunder fluorescent microscopy for lymphatics.

One skilled in the art could easily modify the exemplified studies totest the activity of polynucleotides of the invention (e.g., genetherapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 53 Suppression of TNF Alpha-Induced Adhesion Molecule Expressionby a Polypeptide of the Invention

The recruitment of lymphocytes to areas of inflammation and angiogenesisinvolves specific receptor-ligand interactions between cell surfaceadhesion molecules (CAMs) on lymphocytes and the vascular endothelium.The adhesion process, in both normal and pathological settings, followsa multi-step cascade that involves intercellular adhesion molecule-1(ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and endothelialleukocyte adhesion molecule-1 (E-selectin) expression on endothelialcells (EC). The expression of these molecules and others on the vascularendothelium determines the efficiency with which leukocytes may adhereto the local vasculature and extravasate into the local tissue duringthe development of an inflammatory response. The local concentration ofcytokines and growth factor participate in the modulation of theexpression of these CAMs.

Tumor necrosis factor alpha (TNF-a), a potent proinflammatory cytokine,is a stimulator of all three CAMs on endothelial cells and may beinvolved in a wide variety of inflammatory responses, often resulting ina pathological outcome.

The potential of a polypeptide of the invention to mediate a suppressionof TNF-a induced CAM expression can be examined. A modified ELISA assaywhich uses ECs as a solid phase absorbent is employed to measure theamount of CAM expression on TNF-a treated ECs when co-stimulated with amember of the FGF family of proteins.

To perform the experiment, human umbilical vein endothelial cell (HUVEC)cultures are obtained from pooled cord harvests and maintained in growthmedium (EGM-2; Clonetics, San Diego, Calif.) supplemented with 10% FCSand 1% penicillin/streptomycin in a 37 degree C. humidified incubatorcontaining 5% CO2. HUVECs are seeded in 96-well plates at concentrationsof 1×104 cells/well in EGM medium at 37 degree C. for 18-24 hrs or untilconfluent. The monolayers are subsequently washed 3 times with aserum-free solution of RPMI-1640 supplemented with 100 U/ml penicillinand 100 mg/ml streptomycin, and treated with a given cytokine and/orgrowth factor(s) for 24 h at 37 degree C. Following incubation, thecells are then evaluated for CAM expression.

Human Umbilical Vein Endothelial cells (HUVECs) are grown in a standard96 well plate to confluence. Growth medium is removed from the cells andreplaced with 90 ul of 199 Medium (10% FBS). Samples for testing andpositive or negative controls are added to the plate in triplicate (in10 ul volumes). Plates are incubated at 37 degree C. for either 5 h(selectin and integrin expression) or 24 h (integrin expression only).Plates are aspirated to remove medium and 100 μl of 0.1%paraformaldehyde-PBS (with Ca++ and Mg++) is added to each well. Platesare held at 4° C. for 30 min.

Fixative is then removed from the wells and wells are washed 1× withPBS(+Ca,Mg)+0.5% BSA and drained. Do not allow the wells to dry. Add 10μl of diluted primary antibody to the test and control wells.Anti-ICAM-1-Biotin, Anti-VCAM-1-Biotin and Anti-E-selectin-Biotin areused at a concentration of 10 μg/ml (1:10 dilution of 0.1 mg/ml stockantibody). Cells are incubated at 37° C. for 30 min. in a humidifiedenvironment. Wells are washed ×3 with PBS(+Ca,Mg)+0.5% BSA.

Then add 20 μl of diluted ExtrAvidin-Alkaline Phosphatase (1:5,000dilution) to each well and incubated at 37° C. for 30 min. Wells arewashed ×3 with PBS(+Ca,Mg)+0.5% BSA. 1 tablet of p-Nitrophenol PhosphatepNPP is dissolved in 5 ml of glycine buffer (pH 10.4). 100 μl of pNPPsubstrate in glycine buffer is added to each test well. Standard wellsin triplicate are prepared from the working dilution of theExtrAvidin-Alkaline Phosphatase in glycine buffer: 1:5,000(100)>10-0.5>10-1>10-1.5. 5 μl of each dilution is added to triplicatewells and the resulting AP content in each well is 5.50 ng, 1.74 ng,0.55 ng, 0.18 ng. 100 μl of pNNP reagent must then be added to each ofthe standard wells. The plate must be incubated at 37° C. for 4 h. Avolume of 50 μl of 3M NaOH is added to all wells. The results arequantified on a plate reader at 405 nm. The background subtractionoption is used on blank wells filled with glycine buffer only. Thetemplate is set up to indicate the concentration of AP-conjugate in eachstandard well [5.50 ng; 1.74 ng; 0.55 ng; 0.18 ng]. Results areindicated as amount of bound AP-conjugate in each sample.

Example 54 Method of Creating N- and C-Terminal Deletion MutantsCorresponding to the Human Phosphatase Polypeptides of the PresentInvention

As described elsewhere herein, the present invention encompasses thecreation of N- and C-terminal deletion mutants, in addition to anycombination of N- and C-terminal deletions thereof, corresponding to thehuman phosphatase polypeptides of the present invention. A number ofmethods are available to one skilled in the art for creating suchmutants. Such methods may include a combination of PCR amplification andgene cloning methodology. Although one of skill in the art of molecularbiology, through the use of the teachings provided or referenced herein,and/or otherwise known in the art as standard methods, could readilycreate each deletion mutant of the present invention, exemplary methodsare described below using specific BMY_HPP1, BMY_HPP2, BMY_HPP5 andhuman RET31 deletions as examples.

Briefly, using the isolated cDNA clone encoding the full-length humanBMY_HPP1, BMY_HPP2, BMY_HPP5 or RET31 phosphatase polypeptide sequence(as described elsewhere herein, for example), appropriate primers ofabout 15-25 nucleotides derived from the desired 5′ and 3′ positions ofSEQ ID NO:41, SEQ ID NO:108, SEQ ID NO:149, or SEQ ID NO:151 may bedesigned to PCR amplify, and subsequently clone, the intended N- and/orC-terminal deletion mutant. Such primers could comprise, for example, aninititation and stop codon for the 5′ and 3′ primer, respectively. Suchprimers may also comprise restriction sites to facilitate cloning of thedeletion mutant post amplification. Moreover, the primers may compriseadditional sequences, such as, for example, flag-tag sequences, kozacsequences, or other sequences discussed and/or referenced herein.

For example, in the case of the N9 to L606 BMY_HPP1 N-terminal deletionmutant, the following primers could be used to amplify a cDNA fragmentcorresponding to this deletion mutant:

(SEQ ID NO:167) 5′ 5′-GCAGCA GCGGCCGC AATTTCGGATGGAAGGATTAT Primer GGTG-3′ NotI (SEQ ID NO:168) 3′ 5′- GCAGCA GTCGAC GAGGCCAGGCTTAGGGCCATC -3′Primer SalI

For example, in the case of the M1 to E500 BMY_HPP1 C-terminal deletionmutant, the following primers could be used to amplify a cDNA fragmentcorresponding to this deletion mutant:

(SEQ ID NO:169) 5′ 5′- GCAGCA GCGGCCGC ATGGAGGCTGGCATTTACTT Primer CTAC-3′ NotI (SEQ ID NO:170) 3′ 5′- GCAGCA GTCGAC CACCCAAGACCACATCAAGCPrimer TGC -3′ SalI

For example, in the case of the L31 to K150 BMY_HPP2 N-terminal deletionmutant, the following primers could be used to amplify a cDNA fragmentcorresponding to this deletion mutant:

(SEQ ID NO:171) 5′ Primer 5′-GCAGCA GCGGCCGC CTGTTGGACCTGGGCGTGCGG CACC-3′ NotI (SEQ ID NO:172) 3′ Primer 5′- GCAGCAGTCGAC TTTCGTTCGCTGGTAGAACTGG AAG -3′ SalI

For example, in the case of the M1 to V111 BMY_HPP2 C-terminal deletionmutant, the following primers could be used to amplify a cDNA fragmentcorresponding to this deletion mutant:

(SEQ ID NO:173) 5′ Primer 5′- GCAGCA GCGGCCGC ATGGGCGTGCAGCCCCCCAA CTTC-3′ NotI (SEQ ID NO:174) 3′ Primer 5′- GCAGCAGTCGACCACCAGGTAACAGGCCAGCATG GTG -3′ SalI

For example, in the case of the 1256 to S665 BMY_HPP5 N-terminaldeletion mutant, the following primers could be used to amplify a cDNAfragment corresponding to this deletion mutant:

(SEQ ID NO:104) 5′ Primer 5′-GCAGCA GCGGCCGC ATCGCCTACATCATGAAGAGG ATGG-3′ NotI (SEQ ID NO:105) 3′ Primer 5′- GCAGCAGTCGAC GGAGACCTCAATGATTTCCAT GCTG -3′ SalI

For example, in the case of the M1 to Q367 BMY_HPP5 C-terminal deletionmutant, the following primers could be used to amplify a cDNA fragmentcorresponding to this deletion mutant:

(SEQ ID NO:106) 5′ Primer 5′- GCAGCA GCGGCCGC ATGGCCCATGAGATGATTGG AACTC-3′ NotI (SEQ ID NO:107) 3′ Primer 5′- GCAGCAGTCGAC CTGCACGCTGGGCACGCTGGGC ACG -3′ SalI

For example, in the case of the 1157 to S665 RET31 N-terminal deletionmutant, the following primers could be used to amplify a cDNA fragmentcorresponding to this deletion mutant:

(SEQ ID NO:136) 5′ Primer 5′-GCAGCA GCGGCCGCATGGGCCAACCCGAATTCTT CCC -3′NotI (SEQ ID NO: 137) 3′ Primer 5′- GCAGCA GTCGAC GGAGACCTCAATGATTTCCATGCTG -3′ SalI

For example, in the case of the M1 to K297 RET31 C-terminal deletionmutant, the following primers could be used to amplify a cDNA fragmentcorresponding to this deletion mutant:

(SEQ ID NO:138) 5′ Primer 5′- GCAGCA GCGGCCGC ATGGCCCATGAGATGATTGG AACTC-3′ NotI (SEQ ID NO:139) 3′ Primer 5′- GCAGCAGTCGAC CTTCTTCTCATAGTCCAGGAGT TGG -3′ SalI

For example, in the case of the 1157 to S660 mRET31 N-terminal deletionmutant, the following primers could be used to amplify a cDNA fragmentcorresponding to this deletion mutant:

(SEQ ID NO:140) 5′ Primer 5′-GCAGCA GCGGCCGC ATTGGGCCAACTCGAATTCTT CCC-3′ NotI (SEQ ID NO:141) 3′ Primer 5′- GCAGCAGTCGAC AGAGACCTCGATGATCTCCATG CTG -3′ SalI

For example, in the case of the M1 to T297 mRET31 C-terminal deletionmutant, the following primers could be used to amplify a cDNA fragmentcorresponding to this deletion mutant:

(SEQ ID NO: 142) 5′ Primer 5′- GCAGCA GCGGCCGC ATGGCCCATGAGATGATTGGAACTC -3′ NotI (SEQ ID NO:143) 3′ Primer 5′- GCAGCAGTCGAC CGTCTTCTCATAGTCCATGAGT TGG -3′ SalI

Representative PCR amplification conditions are provided below, althoughthe skilled artisan would appreciate that other conditions may berequired for efficient amplification. A 100 ul PCR reaction mixture maybe prepared using 10 ng of the template DNA (cDNA clone of Humanphosphatase polypeptides), 200 uM 4dNTPs, 1 uM primers, 0.25 U Taq DNApolymerase (PE), and standard Taq DNA polymerase buffer. Typical PCRcycling condition are as follows:

20-25 cycles: 45 sec, 93 degrees  2 min, 50 degrees  2 min, 72 degrees  1 cycle: 10 min, 72 degrees

After the final extension step of PCR, 5 U Klenow Fragment may be addedand incubated for 15 min at 30 degrees.

Upon digestion of the fragment with the NotI and SalI restrictionenzymes, the fragment could be cloned into an appropriate expressionand/or cloning vector which has been similarly digested (e.g., pSport1,among others). The skilled artisan would appreciate that other plasmidscould be equally substituted, and may be desirable in certaincircumstances. The digested fragment and vector are then ligated using aDNA ligase, and then used to transform competent E. coli cells usingmethods provided herein and/or otherwise known in the art.

The 5′ primer sequence for amplifying any additional N-terminal deletionmutants may be determined by reference to the following formula:

(S+(X*3)) to ((S+(X*3))+25), wherein ‘S’ is equal to the nucleotideposition of the initiating start codon of the human BMY_HPP1, BMY_HPP2,BMY_HPP5 or RET31 phosphatase gene (SEQ ID NO:41, SEQ ID NO:149, SEQ IDNO:151, or SEQ ID NO:108, respectively), and ‘X’ is equal to the mostN-terminal amino acid of the intended N-terminal deletion mutant. Thefirst term will provide the start 5′ nucleotide position of the 5′primer, while the second term will provide the end 3′ nucleotideposition of the 5′ primer corresponding to sense strand SEQ ID NO:41,SEQ ID NO:149, SEQ ID NO:151, or SEQ ID NO:108, respectively. Once thecorresponding nucleotide positions of the primer are determined, thefinal nucleotide sequence may be created by the addition of applicablerestriction site sequences to the 5′ end of the sequence, for example.As referenced herein, the addition of other sequences to the 5′ primermay be desired in certain circumstances (e.g., kozac sequences, etc.).

The 3′ primer sequence for amplifying any additional N-terminal deletionmutants may be determined by reference to the following formula:

(S+(X*3)) to ((S+(X*3))-25), wherein ‘S’ is equal to the nucleotideposition of the initiating start codon of the human BMY_HPP1, BMY_HPP2,BMY_HPP5 or RET31 phosphatase genes (SEQ SEQ ID NO:41, SEQ ID NO:149,SEQ ID NO:151, or SEQ ID NO:108, respectively), and ‘X’ is equal to themost C-terminal amino acid of the intended N-terminal deletion mutant.The first term will provide the start 5′ nucleotide position of the 3′primer, while the second term will provide the end 3′ nucleotideposition of the 3′ primer corresponding to the anti-sense strand of SEQSEQ ID NO:41, SEQ ID NO:149, SEQ ID NO:151, or SEQ ID NO:108,respectively. Once the corresponding nucleotide positions of the primerare determined, the final nucleotide sequence may be created by theaddition of applicable restriction site sequences to the 5′ end of thesequence, for example. As referenced herein, the addition of othersequences to the 3′ primer may be desired in certain circumstances(e.g., stop codon sequences, etc.). The skilled artisan would appreciatethat modifications of the above nucleotide positions may be necessaryfor optimizing PCR amplification.

The same general formulas provided above may be used in identifying the5′ and 3′ primer sequences for amplifying any C-terminal deletion mutantof the present invention. Moreover, the same general formulas providedabove may be used in identifying the 5′ and 3′ primer sequences foramplifying any combination of N-terminal and C-terminal deletion mutantof the present invention. The skilled artisan would appreciate thatmodifications of the above nucleotide positions may be necessary foroptimizing PCR amplification.

As mentioned above, the same methodology described for BMY_HPP I,BMY_HPP2, BMY_HPP5 or RET31 N- and C-terminal deletion mutants could beapplied to creating N- and C-terminal deletion mutants corresponding toHPP_BMY1, HPP_BMY2, HPP_BMY3, HPP_BMY4, HPP_BMY5, RET31, and/or mRET31as would be appreciated by the skilled artisan.

One skilled in the art could easily modify the exemplified studies totest the activity of polynucleotides of the invention (e.g., genetherapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 55 Method of Mutating the Human Phosphatases of the PresentInvention Using Site Directed/Site-Specific Mutagenesis

In vitro site-directed mutagenesis is an invaluable technique forstudying protein structure-function relationships and gene expression,for example, as well as for vector modification. Approaches utilizingsingle stranded DNA (ssDNA) as the template have been reported (e.g., T.A. Kunkel et al., 1985, Proc. Natl. Acad. Sci. USA), 82:488-492; M. A.Vandeyar et al., 1988, Gene, 65(1):129-133; M. Sugimoto et al., 1989,Anal. Biochem., 179(2):309-311; and J. W. Taylor et al., 1985, Nuc.Acids. Res., 13(24):8765-8785).

The use of PCR in site-directed mutagenesis accomplishes strandseparation by using a denaturing step to separate the complementarystrands and to allow efficient polymerization of the PCR primers. PCRsite-directed mutagenesis methods thus permit site specific mutations tobe incorporated in virtually any double stranded plasmid, thuseliminating the need for re-subcloning into M13-based bacteriophagevectors or single-stranded rescue. (M. P. Weiner et al., 1995, MolecularBiology: Current Innovations and Future Trends, Eds. A. M. Griffin andH. G. Griffin, Horizon Scientific Press, Norfolk, UK; and C. Papworth etal., 1996, Strategies, 9(3):3-4).

A protocol for performing site-directed mutagenesis, particularlyemploying the QuikChange™ site-directed mutagenesis kit (Stratagene, LaJolla, Calif.; U.S. Pat. Nos. 5,789,166 and 5,923,419) is provided formaking point mutations, to switch or substitute amino acids, and todelete or insert single or multiple amino acids in the RATL1d6 aminoacid sequence of this invention.

Primer Design

For primer design using this protocol, the mutagenic oligonucleotideprimers are designed individually according to the desired mutation. Thefollowing considerations should be made for designing mutagenicprimers: 1) Both of the mutagenic primers must contain the desiredmutation and anneal to the same sequence on opposite strands of theplasmid; 2) Primers should be between 25 and 45 bases in length, and themelting temperature (T_(m)) of the primers should be greater than, orequal to, 78° C. The following formula is commonly used for estimatingthe T_(m) of primers: T=81.5+0.41 (% GC)−675/N−% mismatch. Forcalculating T_(m), N is the primer length in bases; and values for % GCand % mismatch are whole numbers. For calculating T_(m) for primersintended to introduce insertions or deletions, a modified version of theabove formula is employed: T=81.5+0.41 (% GC)−675/N, where N does notinclude the bases which are being inserted or deleted; 3) The desiredmutation (deletion or insertion) should be in the middle of the primerwith approximately 10-15 bases of correct sequence on both sides; 4) Theprimers optimally should have a minimum GC content of 40%, and shouldterminate in one or more C or G bases; 5) Primers need not be5′-phosphorylated, but must be purified either by fast polynucleotideliquid chromatography (FPLC) or by polyacrylamide gel electrophoresis(PAGE). Failure to purify the primers results in a significant decreasein mutation efficiency; and 6) It is important that primer concentrationis in excess. It is suggested to vary the amount of template whilekeeping the concentration of the primers constantly in excess(QuikChange™ Site-Directed Mutagenesis Kit, Stratagene, La Jolla,Calif.).

Protocol for Setting Up the Reactions

Using the above-described primer design, two complimentaryoligonucleotides containing the desired mutation, flanked by unmodifiednucleic acid sequence, are synthesized. The resulting oligonucleotideprimers are purified.

A control reaction is prepared using 5 μl 10× reaction buffer (100 mMKCl; 100 mM (NH₄)₂SO₄; 200 mM Tris-HCl, pH 8.8; 20 mM MgSO₄; 1% Triton®X-100; 1 mg/ml nuclease-free bovine serum albumin, BSA); 2 μl (10 ng) ofpWhitescript™, 4.5-kb control plasmid (5 ng/μl); 1.25 μl (125 ng) ofoligonucleotide control primer #1 (34-mer, 100 ng/μg); 1.25 μl (125 ng)of oligonucleotide control primer #2 (34-mer, 100 ng/μl); 1 μl of dNTPmix; double distilled H₂O; to a final volume of 50 μl. Thereafter, 1 μlof DNA polymerase (PfuTurbo® DNA Polymerase, Stratagene), (2.5 U/μl) isadded. PfuTurbo® DNA Polymerase is stated to have 6-fold higher fidelityin DNA synthesis than does Taq polymerase. To maximize temperaturecycling performance, use of thin-walled test tubes is suggested toensure optimum contact with the heating blocks of the temperaturecycler.

The sample reaction is prepared by combining 5 μl of 10× reactionbuffer; x μl (5-50 ng) of dsDNA template; x μl (125 ng) ofoligonucleotide primer #1; x μl (5-50 ng) of dsDNA template; x μl (125ng) of oligonucleotide primer #2; 1 μl of dNTP mix; and ddH₂O to a finalvolume of 50 μl. Thereafter, 1 μl of DNA polymerase (PfuTurbo DNAPolymerase, Stratagene), (2.5 U/μl) is added.

It is suggested that if the thermal cycler does not have a hot-topassembly, each reaction should be overlaid with approximately 30 μl ofmineral oil.

Cycling the Reactions

Each reaction is cycled using the following cycling parameters:

Segment Cycles Temperature Time 1 1 95° C. 30 seconds 2 12-18 95° C. 30seconds 55° C.  1 minute 68° C.  2 minutes/kb of plasmid length

For the control reaction, a 12-minute extension time is used and thereaction is run for 12 cycles. Segment 2 of the above cycling parametersis adjusted in accordance with the type of mutation desired. Forexample, for point mutations, 12 cycles are used; for single amino acidchanges, 16 cycles are used; and for multiple amino acid deletions orinsertions, 18 cycles are used. Following the temperature cycling, thereaction is placed on ice for 2 minutes to cool the reaction to ≦37° C.

Digesting the Products and Transforming Competent Cells

One μl of the DpnI restriction enzyme (10 U/μl) is added directly (belowmineral oil overlay) to each amplification reaction using a small,pointed pipette tip. The reaction mixture is gently and thoroughly mixedby pipetting the solution up and down several times. The reactionmixture is then centrifuged for 1 minute in a microcentrifuge.Immediately thereafter, each reaction is incubated at 37° C. for 1 hourto digest the parental (i.e., the non-mutated) supercoiled dsDNA.

Competent cells (i.e., XL1-Blue supercompetent cells, Stratagene) arethawed gently on ice. For each control and sample reaction to betransformed, 50 ill of the supercompetent cells are aliquotted to aprechilled test tube (Falcon 2059 polypropylene). Next, 1 μl of theDpnI-digested DNA is transferred from the control and the samplereactions to separate aliquots of the supercompetent cells. Thetransformation reactions are gently swirled to mix and incubated for 30minutes on ice. Thereafter, the transformation reactions are heat-pulsedfor 45 seconds at 42° C. for 2 minutes.

0.5 ml of NZY+ broth, preheated to 42° C. is added to the transformationreactions which are then incubated at 37° C. for 1 hour with shaking at225-250 rpm. An aliquot of each transformation reaction is plated onagar plates containing the appropriate antibiotic for the vector. Forthe mutagenesis and transformation controls, cells are spread onLB-ampicillin agar plates containing 80 μg/ml of X-gal and 20 mM MIPTG.Transformation plates are incubated for >16 hours at 37° C.

Example 56 Complementary Polynucleotides of the BMY_HPP2 Phosphatase ofthe Present Invention

Antisense molecules or nucleic acid sequences complementary to theBMY_HPP2 protein-encoding sequence, or any part thereof, is used todecrease or to inhibit the expression of naturally occurring BMY_HPP2.Although the use of antisense or complementary oligonucleotidescomprising about 15 to 35 base-pairs is described, essentially the sameprocedure is used with smaller or larger nucleic acid sequencefragments. An oligonucleotide based on the coding sequence of BMY_HPP2protein, as shown in FIG. 21, or as depicted in SEQ ID NO:151, forexample, is used to inhibit expression of naturally occurring BMY_HPP2.The complementary oligonucleotide is typically designed from the mostunique 5′ sequence and is used either to inhibit transcription bypreventing promoter binding to the coding sequence, or to inhibittranslation by preventing the ribosome from binding to the BMY_HPP2protein-encoding transcript. However, other regions may also betargeted.

Using an appropriate portion of the signal and/or 5′ sequence of SEQ IDNO:151, an effective antisense oligonucleotide includes any of about15-35 nucleotides spanning the region which translates into the signalor 5′ coding sequence, among other regions, of the polypeptide as shownin FIG. 21 (SEQ ID NO:152). Appropriate oligonucleotides are designedusing OLIGO 4.06 software and the BMY_HPP2 protein coding sequence (SEQID NO:151). Preferred oligonucleotides are dideoxy based and areprovided below. The oligonucleotides were synthesized using chemistryessentially as described in U.S. Pat. No. 5,849,902; which is herebyincorporated herein by reference in its entirety.

ID# Sequence 13600 GGAUAUCACUACUGCAUUGCCUGGA (SEQ ID NO:179) 13601UACAGCAGAUCUGUGCAGGCCAGGU (SEQ ID NO:180) 13602UGAUCACACAGUAGCGGAAGAUGCU (SEQ ID NO:181) 13603AGGAGUAGCAGAAUGGUUAGCCUUC (SEQ ID NO:182) 13604UGAAAGCAGGCGAGAUUCGAUCCGA (SEQ ID NO:183)

The BMY_HPP2 polypeptide has been shown to be involved in the regulationof the mammalian cell cycle. Subjecting cells with an effective amountof a pool of all five of the above antisense oligoncleotides resulted ina significant increase in Cyclin D expression/activity providingconvincing evidence that BMY_HPP2 at least regulates the activity and/orexpression of Cyclin D either directly, or indirectly. Moreover, theresults suggest the physiological role of BMY_HPP2 is the negativeregulation of Cyclin D activity and/or expression, either directly orindirectly. The Cyclin D assay used is described below and was basedupon the analysis of Cyclin D activity as a downstream marker forproliferative signal transduction events.

Transfection of Post-Quiescent A549 Cells with AntiSenseOligonucleotides.

Materials needed:

-   -   A549 cells maintained in DMEM with high glucose (Gibco-BRL)        supplemented with 10% Fetal Bovine Serum, 2 mM L-Glutamine, and        1× penicillin/streptomycin.    -   Opti-MEM (Gibco-BRL)    -   Lipofectamine 2000 (Invitrogen)    -   Antisense oligomers (Sequitur)    -   Polystyrene tubes.    -   Tissue culture treated plates.

Quiescent cells were prepared as follows:

-   Day 0: 300,000 A549 cells were seeded in a T75 tissue culture flask    in 10 ml of A549 media, and incubated in at 37° C., 5% CO₂ in a    humidified incubator for 48 hours.-   Day 2: The T75 flasks were rocked to remove any loosely adherent    cells, and the A549 growth media removed and replenished with 10 ml    of fresh A549 media. The cells were cultured for six days without    changing the media to create a quiescent cell population.-   Day 8: Quiescent cells were plated in multi-well format and    transfected with antisense oligonucleotides.

A549 cells were transfected according to the following:

-   -   1. Trypsinize T75 flask containing quiescent population of A549        cells.    -   2. Count the cells and seed 24-well plates with 60K quiescent        A549 cells per well.    -   3. Allow the cells to adhere to the tissue culture plate        (approximately 4 hours).    -   4. Transfect the cells with antisense and control        oligonucleotides according to the following:        -   a. A 10× stock of lipofectamine 2000 (10 ug/ml is 10×) was            prepared, and diluted lipid was allowed to stand at RT for            15 minutes. Stock solution of lipofectamine 2000 was 1            mg/ml. 10× solution for transfection was 10 ug/ml. To            prepare 10× solution, dilute 10 ul of lipofectamine 2000            stock per 1 ml of Opti-MEM (serum free media).        -   b. A 10× stock of each oligomer was prepared to be used in            the transfection. Stock solutions of oligomers were at 100            uM in 20 mM HEPES, pH 7.5. 10× concentration of oligomer was            0.25 uM. To prepare the 10× solutions, dilute 2.5 ul of            oligomer per 1 ml of Opti-MEM.        -   c. Equal volumes of the 10× lipofectamine 2000 stock and the            10× oligomer solutions were mixed well, and incubated for 15            minutes at RT to allow complexation of the oligomer and            lipid. The resulting mixture was 5×.        -   d. After the 15 minute complexation, 4 volumes of full            growth media was added to the oligomer/lipid complexes            (solution was 1×).        -   e. The media was aspirated from the cells, and 0.5 ml of the            1× oligomer/lipid complexes added to each well.        -   f. The cells were incubated for 16-24 hours at 37° C. in a            humidified CO₂ incubator.        -   g. Cell pellets were harvested for RNA isolation and TaqMan            analysis of downstream marker genes.            TaqMan Reactions

Quantitative RT-PCR analysis was performed on total RNA preps that hadbeen treated with DNaseI or poly A selected RNA. The Dnase treatment maybe performed using methods known in the art, though preferably using aQiagen Rneasy kit to purify the RNA samples, wherein DNAse I treatmentis performed on the column.

Briefly, a master mix of reagents was prepared according to thefollowing table:

Dnase I Treatment Reagent Per r'xn (in uL) 10x Buffer 2.5 Dnase I (1unit/ul @ 1 unit per ug 2 sample) DEPC H₂O 0.5 RNA sample @ 0.1 ug/ul 20(2-3 ug total) Total 25

Next, 5 ul of master mix was aliquoted per well of a 96-well PCRreaction plate (PE part # N801-0560). RNA samples were adjusted to 0.1ug/ul with DEPC treated H₂O (if necessary), and 20 ul was added to thealiquoted master mix for a final reaction volume of 25 ul.

The wells were capped using strip well caps (PE part # N801-0935),placed in a plate, and briefly spun in a centrifuge to collect allvolume in the bottom of the tubes. Generally, a short spin up to 500 rpmin a Sorvall RT is sufficient

The plates were incubated at 37° C. for 30 mins. Then, an equal volumeof 0.1 mM EDTA in 10 mM Tris was added to each well, and heatinactivated at 70° C. for 5 min. The plates were stored at −80° C. uponcompletion.

RT Reaction

A master mix of reagents was prepared according to the following table:

RT reaction RT No RT Reagent Per Rx'n (in ul) Per Rx'n (in ul) 10x RTbuffer 5 2.5 MgCl₂ 11 5.5 DNTP mixture 10 5 Random Hexamers 2.5 1.25Rnase inhibitors 1.25 0.625 RT enzyme 1.25 — Total RNA 500 ng (100 ng19.0 max 10.125 max no RT) DEPC H₂O — — Total 50 uL 25 uL

Samples were adjusted to a concentration so that 500 ng of RNA was addedto each RT rx′n (10 ng for the no RT). A maximum of 19 ul can be addedto the RT rx′n mixture (10.125 ul for the no RT.) Any remaining volumeup to the maximum values was filled with DEPC treated H₂O, so that thetotal reaction volume was 50 ul (RT) or 25 ul (no RT).

On a 96-well PCR reaction plate (PE part # N801-0560), 37.5 ul of mastermix was aliquoted (22.5 ul of no RT master mix), and the RNA sampleadded for a total reaction volume of 50 ul (25 ul, no RT). Controlsamples were loaded into two or even three different wells in order tohave enough template for generation of a standard curve.

The wells were capped using strip well caps (PE part # N801-0935),placed in a plate, and spin briefly in a centrifuge to collect allvolume in the bottom of the tubes. Generally, a short spin up to 500 rpmin a Sorvall RT is sufficient.

For the RT-PCR reaction, the following thermal profile was used:

-   -   25° C. for 10 min    -   48° C. for 30 min    -   95° C. for 5 min    -   4° C. hold (for 1 hour)    -   Store plate @−20° C. or lower upon completion.        TaqMan Reaction (Template Comes from RT Plate.)

A master mix was prepared according to the following table:

TaqMan reaction (per well) Reagent Per Rx'n (in ul) TaqMan Master Mix4.17 100 uM Probe .025 (SEQ ID NO: 186) 100 uM Forward .05 primer (SEQID NO: 184) 100 uM Reverse .05 primer (SEQ ID NO: 185) Template — DEPCH₂O 18.21 Total 22.5The primers used for the RT-PCR reaction is as follows:Cyclin D Primer and Probes:

Forward Primer: ACTACCGCCTCACACGCTTC (SEQ ID NO:184) Reverse Primer:CTTGACTCCAGCAGGGCTTC (SEQ ID NO:185) TaqMan Probe:ATCAAGTGTGACCCAGACTGCCTCCG (SEQ ID NO:186)

Using a Gilson P-10 repeat pipetter, 22.5 ul of master mix wasaliquouted per well of a 96-well optical plate. Then, using P-10pipetter, 2.5 ul of sample was added to individual wells. Generally, RTsamples are run in triplicate with each primer/probe set used, and no RTsamples are run once and only with one primer/probe set, often gapdh (orother internal control).

A standard curve is then constructed and loaded onto the plate. Thecurve has five points plus one no template control (NTC, =DEPC treatedH₂O). The curve was made with a high point of 50 ng of sample (twice theamount of RNA in unknowns), and successive samples of 25, 10, 5, and 1ng. The curve was made from a control sample(s) (see above).

The wells were capped using optical strip well caps (PE part #N801-0935), placed in a plate, and spun in a centrifuge to collect allvolume in the bottom of the tubes. Generally, a short spin up to 500 rpmin a Sorvall RT is sufficient.

Plates were loaded onto a PE 5700 sequence detector making sure theplate is aligned properly with the notch in the upper right hand corner.The lid was tightened down and run using the 5700 and 5700 quantitationprogrames and the SYBR probe using the following thermal profile:

-   -   50° C. for 2 min    -   95° C. for 10 min    -   and the following for 40 cycles:        -   95° C. for 15 sec        -   60° C. for 1 min    -   Change the reaction volume to 25 ul.

Once the reaction was complete, a manual threshold of around 0.1 was setto minimuze the background signal. Additional information relative tooperation of the GeneAmp 5700 machine may be found in reference to thefollowing manuals: “GeneAmp 5700 Sequence Detection System OperatorTraining CD”; and the “User's Manual for 5700 Sequence DetectionSystem”; available from Perkin-Elmer and hereby incorporated byreference herein in their entirety.

Cyclin D1 is a critical regulator of the process of cell division. Ithas been identified as an early modulator of the G1 phase of the cellcycle, and cyclin D1 expression increases as cells enter that phase ofthe cell cycle. It has long been thought that an ability topharmacologically block cancerous cells in any part of the cell cyclewill have a negative impact on the tumor and be beneficial for managingthe disease. Support for this rationale comes from the observation thateffective drugs such as Taxol block the cell cycle in G2 phase.Importantly, the rapidly dividing cells found in the cancerous staterequire abundant levels of cyclin D1 to maintain an accelerated rate ofproliferation and proceed to S-phase. Most noteably, overexpression ofcyclinD1 is a hallmark of several types of human tumors, especiallybreast tumors (J Mammary Gland Biol Neoplasia 1996 April; 1(2):153-62).As such, it is thought that drugs that affect cyclin D1, directly orindirectly, would block cancer cells from dividing and have a beneficialeffect for patients. Such drug targets could lie within the signaltransduction pathway between the oncogene ras and the nucleus, wherecell cycle modulators control DNA synthesis (J. Biol Chem 2000, Oct. 20;275 (42):32649-57). Even more evidence exists suggesting that the Wntpathway, mediated by the tumor suppressor betacatenin, regulates thecell cycle via transcriptional control of cyclinD1 (Oncogene 2001 Aug.23; 20(37):5093-9: PNAS 2000 Apr. 11; 97(8):4262-6). Thus targetsinfluencing beta catenin/TCF4 function could also affect cyclin D1transcript levels. As mutations in oncogenes such as ras, and tumorsuppressors such as beta catenin are common to may cancers, it isobvious that cyclinD1 levels are indicative of the condition of the celland its preparedness to proliferate, and affecting cyclinD1 levels andactivity could be achieved by numerous mechanisms embodied in multiplepathways.

Antisense inhibition of the HPP_BMY2 phosphatase levels provokes aresponse in A549 cells that indicates the regulatory pathwayscontrolling cydlinD1 levels are affected. This implicates HPP_BMY2 inpathways important for maintenance of the proliferative state andprogression through the cell cycle. As stated above, there are numerouspathways that could have either indirect or direct affects on thetranscriptional levels of cyclin D1. Importantly, a major part of thepathways implicated involve the regulation of protein activity throughphosphorylation. In as much as HPP_BMY2 is a phosphatase enzyme, it isreadily conceivable that dephosphorylation of proteins, the counteractivity to the kinases in the signal transduction cascades, contributesto the signals determining cell cycle regulation and proliferation,including regulating cyclin D1 levels. Additionally, the complexity ofthe interactions between proteins in the pathways described also allowfor affects on the pathway eliciting compensatory responses. That is,inhibition of one pathway affecting cyclinD1 activity could provoke amore potent response and signal from another pathway of the same end,resulting in upregulation of cyclin D1. Thus, the effect of inhibitionof HPP_BMY2 resulting in slight increases in cyclin D1 levels couldindicate that one pathway important to cancer is effected in a way toimplicate HPP_BMY2 as a potential target for pharmacologic inhibitionfor cancer treatment, yet a parallel pathway in the context of theexperiment would replace HPP_BMY2 and propagate dysregulation of CyclinD1.

Example 57 Method of Creating RET31 and Truncated RET31 Fusion ProteinConstructs and Methods of Expression and Purification of the Same

The GST fusion proteins were designed to contain the full-length RET31protein sequence (SEQ ID NO:109), as well as a C-terminal deletionmutant of the RET31 protein sequence corresponding to amino acids M1 toT302 of SEQ ID NO:109 which was truncated after the phosphatase homologydomain ending at about amino acid residue 297 of SEQ ID NO:109.

In order to generate the RET31 fusion proteins, three PCR primers weredesigned and received from Life Technologies (Gaithersburg, Md.). Theoligos were:

Oligo number Name Sequence S5972B08 RET31for5′-CATATGGGATCCATGGCCCATGAGATTG (SEQ ID NO: 187) S5972B09 RET31rev5′-GGTACCCTCGAGTCAGGAGACCTCAAT GAT (SEQ ID NO:188) S6311A01 RET31rev2-25′-GGTACCCTCGAGTCAAGTCTGGTTCTT AAT (SEQ ID NO:189)

Clones containing the original gene sequence of the full-length RET 31polynucleotide (SEQ ID NO:108) were used as a template for thesubsequent PCR. The clone was linearized using a restriction enzymeprior to PCR. PCR was performed using random hexamers and the ExpandHigh Fidelity PCR System (ROCHE). Amplification was achieved using RET31forward primer (SEQ ID NO:187) paired with either RET31 Rev (SEQ IDNO:188) or Rev2-2 (SEQ ID NO:189), for the full-length cDNA or truncatedcDNA respectively. The thermocycler settings were 94° C. for 30 sec, 55°C. for 30 sec and 72° C. for 60 seconds for 25 cycles. The amplimerswere gel purified by the QIAgen Extraction kit (QIAgen, Valencia,Calif.) and ligated, using T4 DNA ligase, into the pGEX 4T3 Vector(Amersham Pharmacia Biotech) and sequenced using standard methods.

Appropriate clones were chosen based upon the sequencing data, and wereused for subsequent steps. Protein expression was induced with 0.1 mMIPTG over a 5-hour period. The fusion protein was isolated following themethods outlined in Ausubel, et al., 1992, Short Protocols in MolecularBiology, John Wiley and Sons, Inc., pp. 16-28 to 16-31, using GST beads(Pierce) and reduced Glutathione (Sigma). The predicted proteins wereapproximately 100 kD for the full-length protein and 60 kD for thetruncated protein. To confirm that GST fusion protein was present, theproteins, along with appropriate markers, were run on a 4-12% NuPage BISTRIS Gel with Mops buffer and transferred to a PVD membrane at 4° C. Themembrane was blocked with 5% nonfat dry milk in TBS, and probed with arabbit anti-GST antibody (developed in house). A goat anti-rabbitconjugated to HRP secondary antibody (Biorad) was used and the blot wasdeveloped with ECL reagent (Amersham Pharmacia Biotech)—data not shown.

Example 57 Method of Assaying the Phosphatase Activity of the RET31Polypeptide

The phosphatase activity for the full-length RET31 and the M1 to T302C-terminal RET31 GST fusion proteins were measured by assaying theability of the proteins to hydrolyze para-nitrophenylphosphate, acompound known to be a substrate for phosphatases, as described inKrejsa, C. et al., J. Biol. Chem. Vol. 272, p. 11541-11549, 1997 (whichis hereby incorporated in its entirety herein). The proteins areincubated with para-nitrophenylphosphate in a solution containing 10 mMimidazole, pH 7.0, 1 mM EDTA, 2 mM dithiothreitol, and 5 μg/ml BSA for 2hours with and without sodium orthovanadate (Fisher) prepared indistilled water. The progress of the phosphatase reaction in a 96-wellformat was monitored by the OD405 nm on a plate reader (MolecularDevices) at 10-minute intervals in the kinetic mode.

The RET31-GST full length (FL), M1 to T302 C-terminal RET31-GST (trunc),or GST alone were purified and assayed for cleavage ofpara-nitrophenylphosphate (pNPP). The bars represent the average oftriplicate determinations, and the standard deviations are as shown.Each protein preparation was assayed in the absence and presence of 2 mMof the phosphatase inhibitor orthovanadate. The full length andtruncated versions clearly demonstrated activity compared to the GSTprotein as shown in FIG. 36. In addition, the full length and truncatedprotein phosphatase activity was blocked by the phosphatase inhibitorvanadate, as shown.

Of particular significance is the unexpected five fold increase inphosphatase activity of the M1 to T302 C-terminal RET31-GST (trunc)fusion protein relative to the RET31-GST full length (FL) fusionprotein.

While the described phosphatase assay elucidated the phosphataseactivity of the full-length RET31 (SEQ ID NO:109) and M1 to T302 RET31C-terminal deletion mutant (amino acids 1 to 302 of SEQ ID NO:109),subsequent sequencing of the RET31-GST full length (FL) and M1 to T302C-terminal RET31-GST (trunc) fusion protein constructs determined thatseveral amino acid mutations were unintentionally introduced duringtheir construction. The sequences of the RET31 portions of both fusionproteins are provided below. Since the location of these mutations arenot within the conserved phosphatase domain nor near any active siteresidues, it is not believed they would have any effect on thephosphatase activity of either construct. Rather, the observedphosphatase activity is believed to be representative of the wild typeRET31 polypeptide sequence (SEQ ID NO:109) for the RET3′-GST full length(FL), while the observed phosphatase activity of the M1 to T302C-terminal RET31-GST (trunc) fusion protein is believed to berepresentative of the wild type M1 to T302 C-terminal RET31 C-terminaldeletion (amino acids M1 to T302 of SEQ ID NO:109). One skilled in theart of molecular biology could easily correct the mutations of bothconstructs using known methods in conjunction with the information andteachings described herein. Nonetheless, the polypeptide sequences ofthe RET31 portion of both fusion proteins are encompassed by the presentinvention.

In preferred embodiments, the following RET31 polypeptide is encompassedby the present invention:MAHEIGTQIVTERLVALLESGTEKVLLIDSRPFVEYNTSHILEAININCSKLMKRRLQQDKVLITELIQHSAKHKVDIDCSQKVVVYDQSSQDVASLSSDCFLTVLLGKLEKSFNSVHLLAGGFAEFSRCFPGLCEGKSTLVPTCISQPCLPVANIGPTRILPNLYLGCQRDVLNKELMQQNGIGYVLNASNTCPKPDFIPESHFLRVPVNDSFCEKILPWLDKSVDFIEKAKASNGCVLVHCLAGISRSATIAIAYIMKRMDMSLDEAYRFVKEKRPTISPSFNFLGQLLDYEKKIKNQAGASGPKS KLKLLHLEKPNEPVPAVSEGGQKSETPLSPPCADSATSEAAGQRPVHPASVPSVPSVQPSLLEDSPLVQALSGLHLSADRLEDSNKLKRSFSLDIKSVSYSASMAASLHGFSSSEDALEYYKPSTTLDGTNKLCQFSPVQELSEQTPETSPDKEEASIPK KLQTARPSDSQSKRLHSVRTSSSGTAQRSLLSPLHRSGSVEDNYHTSFLFGLSTSQQHLTKSAGLGLKGWHSDILAPQTSTPSLTSSWYFATESSHFYSASAIYGGSASYSAYSRSQLPTCGDQVYSVRRRQKPSDRADSRRSWHEESPFEKQFKRRSCQMEFGESIMSENRSREELGKVGSQSSFSGSMEIEVS (SEQ ID NO:190). Polynucleotides encoding thispolypeptide are also provided.

In preferred embodiments, the following M1 to T302 RET31 polypeptide isencompassed by the present invention:MAHEIVGTQIVTERLVALLESGTEKVLLIDSRPFVEYNTSHILEAININCSKLMKRRLQQDKVLITELIQHSAKHKVDIDCSQKVVVYDQSSQDVASLSSDCFLTVLLGKLEKSFNSVHLLAGGFAEFSRCFPGLCEGKSTLVPTCISQPCLPVANIGPTRILPNLYLGCQRDVLNKELMQQNGIGYVLNASNTCPKPDFIPESHFLRVPVNDSFCEKILPWLDKSVDFIEKAKASNGCVLVHCLAGISRSATIAIAYIMKRMDMSLDEAYRFVKEKRPTISPSFNFLGQLL DYEKKIKNQT(SEQ ID NO:191). Polynucleotides encoding this polypeptide are alsoprovided.

The present invention encompasses the application of this phosphataseactivity assay to the other phosphatases of the present invention.

Example 58 Method of Assessing the Expression Profile of the RET31Phosphatase Polypeptides of the Present Invention at the Level of theProtein Using Immunohistochemistry

Peptide Selection and Antibody Production

The sequence for the RET31 polypeptide (SEQ ID NO:109) was analyzed bythe algorithm of Hopp and Woods to determine potential peptides forsynthesis and antibody production. The peptides were then BLASTedagainst the SWISS-PROT database to determine the uniqueness of theidentified peptide and to help predict the specificity of the resultingantibodies. The following RET31 polypeptide fragments were selectedaccording to the methods above for peptide synthesis:KNQTGASGPKSKKLKLLHLE (SEQ ID NO:192); and CKKLQTARPSDSQSKRLHS (SEQ IDNO:193). Rabbit polyclonal antisera was generated for both synthesizedRET31 peptides. In order to allow for peptide conjugation to the carrierprotein, a cysteine residue was added to the N-terminus of the SEQ IDNO:193 peptide. The third bleeds were subjected to peptide affinitypurification, and the resulting antisera were then used as primaryantibodies in immunohistochemistry experiments. The antisera for the SEQID NO:192 peptide was labeled RET31 antibody 299, while the antisera forthe SEQ ID NO:193 peptide was labeled RET31 antibody 469 antibody.

Antibody Titration Protocol and Positive Control Study Results

Antibody titration experiments were conducted with RET31 antibodies 299and 469 (both rabbit polyclonals) to establish concentrations that wouldresult in minimal background and maximal detection of signal. Serialdilutions were performed at 1:50. 1:100, 1:250, 1:500, and 1:1000. Theserial dilution study demonstrated the highest signal-to-noise ratios atdilutions 1:250 and 1:400, on paraffin-embedded, formalin-fixed tissuesfor both antibodies. These concentrations were used for the study. RET31antibodies 299 and 469 were used as primary antibodies, and theprincipal detection system consisted of a Vector anti-rabbit secondary(BA-1000; DAKO Corp.), a Vector ABC-AP Kit (AK-5000; DAKO Corp.) with aVector Red substrate kit (SK-5100; DAKO Corp.), which was used toproduce a fuchsia-colored deposit. Tissues were also stained with apositive control antibody (CD31) to ensure that the tissue antigens werepreserved and accessible for immunohistochemical analysis. Only tissuesthat stained positive for CD31 were chosen for the remainder of thestudy. The negative control consisted of performing the entireimmunohistochemistry procedure on adjacent sections in the absence ofprimary antibody. Slides were imaged using a DVC 1310C digital cameracoupled to a Nikon microscope. Images were stored as TIFF files usingAdobe PhotoShop.

Immunohistochemistry Procedure

Slides containing paraffin sections (LifeSpan BioSciences, Inc.;Seattle, Wash.) were deparaffinized through xylene and alcohol,rehydrated, and then subjected to the steam method of target retrieval(#S1700; DAKO Corp.; Carpenteria, Calif.). Immunohistochemical assaytechniques are commonly known in the art and are described brieflyherein. Immunocytochemical (ICC) experiments were performed on a DAKOautostainer following the procedures and reagents developed by DAKO.Specifically, the slides were blocked with avidin, rinsed, blocked withbiotin, rinsed, protein blocked with DAKO universal protein block,machine blown dry, primary antibody, incubated, and the slides rinsed.Biotinylated secondary antibody was applied using the manufacturer'sinstructions (1 drop/10 ml, or approximately 0.75 μg/mL), incubated,rinsed slides, and applied Vectastain ABC-AP reagent for 30 minutes.Vector Red was used as substrate and prepared according to themanufacturer's instructions just prior to use.

Immunohistochemistry Results

The immunohistochemistry results were consistent with the Northern Blotand RT-PCR expression profiles described elsewhere herein for the RET31polypetide. Specifically, moderate to strong staining was observed innormal respiratory epithelial cell bodies and cilia. Types I and IIpneumocytes were also moderately positive, as were neutrophils, mastcells, and macrophages in normal lung. In asthmatic patients,respiratory epithelial cell bodies stained less intensely, but ciliacontinued to stain strongly. Pneumocytes also stained less intenselythan normal tissue. Inflammatory cell staining did not differ fromnormal tissue. Bronchial smooth muscle stained faintly in normal andasthmatic lungs. Cytoplasmic, diffuse nucleoplasmic, and nucleolarstaining was observed in several cell types, including vascularendothelial and respiratory epithelial cells.

Moderate to strong staining was seen in chondrocytes and rimmingosteoblasts in degenerative arthritis. In constrast, osteocytes werenegative, as was the osteoid matrix. Hematopoetic tissue showed stronglypositive cytoplasm and nucleus in myeloid series cells at all stages ofmaturation. Megakaryocytic and erythroid cells were negative.

Schwann cells and vascular endothelial cells were moderately to stronglypositive in normal colon, in contrast to epithelial cells and ganglioncells, which were negative. Inflammatory cells, such as neutrophils,eosinophils, macrophages, and mast wells were strongly positive. Plasmacells showed blush to faint staining. Lymphocytes in norml colon showedstrong punctate nuclear and nucleolar staining. In contrast to normalcolon, the colon sections with ulcerative colitis showed less prominentnucleolar staining in lymphocytes. Neuroendocrine cells in theepithelium were faintly positive.

Normal lung showed strong cilial staining in the respiratory epithelialcells, with only blush, diffuse, nuclear staining in the cell body ofthese cells. Pneumocytes were faintly to moderately positive, as werealveolar macrophages and vascular endothelium. Asthmatic lungs continuedto show strong cilial staining, but showed blush positivity in normallung, and were predominately negative in diseased lung. Pneumocytestaining varied from blush to moderately positive in asthmatic lungs.Pneumocyte staining wa unchanged from normal lung. Inflammatory cellstaining was similar to normal tissue.

Moderate staining was seen in the stratum granulosum in normal skin,whereas the other layers were negative or showed blush positivity.Melanocytes were moderately to strongly positive, as were hair folliclesand eccrine and sebaceous glands. Skin with psoriasis showed strongstaining in the stratum granulosum, increased from normal skin. Incontrast to normal skin, melanocytes in skin were negative. In thepsoriasisform dermatitis sample, the staining pattern was similar tothat observed in normal skin.

In synovium, the reactive synoviocytes in one sample of rheumatoidarthritis were faintly to moderately positive, in contrast to normalsynoviocytes, which were negative or showed blush staining. In thesecond sample of rheumatoid arthritis, the difference in synoviocytestaining was smaller than in the first sample.

Interesting observations in this study included the very prominentstaining of the nucleolus of lymphocytes and other cell types. Ininflammatory bowel disease, the lymphocytes did not show nucleolarstaining as prominately as in normal colon. Skin with psorisis had veryprominent staining of the stratum granulosum, in comparison to normalskin or to the psoriasiform dermatitis sample.

The present invention encompasses the application of this phosphataseactivity assay to the other phosphatases of the present invention.

Example 59 Method of Assessing the Expression Profile of the NovelPhosphatases of Polypeptides of the Present Invention Using ExpandedmRNA Tissue and Cell Sources

Total RNA from tissues was isolated using the TriZol protocol(Invitrogen) and quantified by determining its absorbance at 260 nM. Anassessment of the 18s and 28s ribosomal RNA bands was made by denaturinggel electrophoresis to determine RNA integrity.

The specific sequence to be measured was aligned with related genesfound in GenBank to identity regions of significant sequence divergenceto maximize primer and probe specificity. Gene-specific primers andprobes were designed using the ABI primer express software to amplifysmall amplicons (150 base pairs or less) to maximize the likelihood thatthe primers function at 100% efficiency. All primer/probe sequences weresearched against Public Genbank databases to ensure target specificity.Primers and probes were obtained from ABI.

For BMY_HPP1, the primer probe sequences were as follows

Forward Primer 5′-TCAGAGAATGGGCCAACAAGA-3′ (SEQ ID NO:194) ReversePrimer 5′-CGAAAACGCTCGAGGAATGA-3′ (SEQ ID NO:195) TaqMan Probe5′ -CAGGCCTAGGTTCCTCCTCTCGGAAA-3′ (SEQ ID NO:196)For BMY_HPP2, the primer probe sequences were as follows

Forward Primer 5′-TCAGAGAATGGGCCAACAAGA-3′ (SEQ ID NO: 197) ReversePrimer 5′-CGAAAACGCTCGAGGAATGA-3′ (SEQ ID NO:198) TaqMan Probe5′-CAGGCCTAGGTTCCTCCTCTCGGAAA-3′ (SEQ ID NO:199)For BMY_HPP4, the primer probe sequences were as follows

Forward Primer 5′-TCAGAGAATGGGCCAACAAGA-3′ (SEQ ID NO:200) ReversePrimer 5′-CGAAAACGCTCGAGGAATGA-3′ (SEQ ID NO:201) TaqMan Probe5′ -CAGGCCTAGGTTCCTCCTCTCGGAAA-3′ (SEQ ID NO:202)For BMY_HPP5 (RET31), the primer probe sequences were as follows

Forward Primer 5′-TCAGAGAATGGGCCAACAAGA-3′ (SEQ ID NO:203) ReversePrimer 5′-CGAAAACGCTCGAGGAATGA-3′ (SEQ ID NO:204) TaqMan Probe5′ -CAGGCCTAGGTTCCTCCTCTCGGAAA-3′ (SEQ ID NO:205)

The same BMY_HPP5 primer probe sequences hybridize to the RET31 mRNAsequences as well. Therefore, the expression profiling for BMY_HPP5 isalso representative of the RET31 expression profile as well.

DNA Contamination

To access the level of contaminating genomic DNA in the RNA, the RNA wasdivided into 2 aliquots and one half was treated with Rnase-free Dnase(Invitrogen). Samples from both the Dnase-treated and non-treated werethen subjected to reverse transcription reactions with (RT+) and without(RT−) the presence of reverse transcriptase. TaqMan assays were carriedout with gene-specific primers (see above) and the contribution ofgenomic DNA to the signal detected was evaluated by comparing thethreshold cycles obtained with the RT+/RT− non-Dnase treated RNA to thaton the RT+/RT− Dnase treated RNA. The amount of signal contributed bygenomic DNA in the Dnased RT− RNA must be less that 10% of that obtainedwith Dnased RT+ RNA. If not the RNA was not used in actual experiments.

Reverse Transcription Reaction and Sequence Detection

100 ng of Dnase-treated total RNA was annealed to 2.5 μM of therespective gene-specific reverse primer in the presence of 5.5 mMMagnesium Chloride by heating the sample to 72° C. for 2 min and thencooling to 55° C. for 30 min. 1.25 U/μl of MuLv reverse transcriptaseand 500M of each dNTP was added to the reaction and the tube wasincubated at 37° C. for 30 min. The sample was then heated to 90° C. for5 min to denature enzyme.

Quantitative sequence detection was carried out on an ABI PRISM 7700 byadding to the reverse transcribed reaction 2.5 μM forward and reverseprimers, 500 μM of each dNTP, buffer and 5 U AmpliTaq Gold™. The PCRreaction was then held at 94° C. for 12 min, followed by 40 cycles of94° C. for 15 sec and 60° C. for 30 sec.

Data Handling

The threshold cycle (Ct) of the lowest expressing tissue (the highest Ctvalue) was used as the baseline of expression and all other tissues wereexpressed as the relative abundance to that tissue by calculating thedifference in Ct value between the baseline and the other tissues andusing it as the exponent in 2^((ΔCt))

mRNA levels were assayed in samples from three individual donors foreach tissue for each human phosphatase polypeptide. Values presentedrepresent the average abundance of each human phosphatase polypeptidefor each tissue divided by the average abundance of said polypeptide inthe tissue with the lowest level of expression. For example, the lowestexpression level detected for each polypeptide is as follows:BMY_HPP1=blood mononuclear cells; BMY_HPP2=umbilical cord;BMY_HPP4=blood mononuclear cells; and BMY_HPP5 (RET31)=umbilical cord.The expanded expression profile of BMY_HPP1, BMY_HPP2, BMY_HPP4, andBMY_HPP5 (RET31), are provided in FIGS. 26, 30, 34, and 35 and aredescribed elsewhere herein.

It will be clear that the invention may be practiced otherwise than asparticularly described in the foregoing description and examples.Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, are within thescope of the appended claims.

The entire disclosure of each document cited (including patents, patentapplications, journal articles, abstracts, laboratory manuals, books, orother disclosures) in the Background of the Invention, DetailedDescription, and Examples is hereby incorporated herein by reference.Further, the hard copy of the sequence listing submitted herewith andthe corresponding computer readable form are both incorporated herein byreference in their entireties.

TABLE III GENBANK ACCESSION NO: Q9ZSE4 SERINE/THREONINE PROTEINPHOSPHATASE TYPE 2A. GENBANK ACCESSION NO: Q16341 PROTEIN-TYROSINEPHOSPHATASE. GENBANK ACCESSION NO: P2C2_CAEEL PROBABLE PROTEINPHOSPHATASE 2C T23F11.1 (EC 3.1.3.16) (PP2C). GENBANK ACCESSION NO:Q92140 PROTEIN PHOSPHATASE 2A, CATALYTIC SUBUNIT, BETA ISOFORM. GENBANKACCESSION NO: Q28006 BA14 TYROSINE PHOSPHATASE (EC 3.1.3.48). GENBANKACCESSION NO: O14428 SERINE/THREONINE PROTEIN PHOSPHATASE PPT1. GENBANKACCESSION NO: P2CG_MOUSE PROTEIN PHOSPHATASE 2C GAMMA ISOFORM (EC3.1.3.16) (PP2C-GAMMA) (PROTEIN PHOSPHATASE 1C) (FIBROBLAST GROWTHFACTOR INDUCIBLE PROTEIN 13) (FIN13). GENBANK ACCESSION NO: Q64604PROTEIN-TYROSINE PHOSPHATASE, RECEPTOR-TYPE, F POLYPEPTIDE PRECURSOR (EC3.1.3.48) (LAR PROTEIN) (LEUKOCYTE ANTIGEN RELATED) (LEUKOCYTE COMMONANTIGEN-RELATED PHOSPHATASE) (PROTEIN-TYROSINE-PHOSPHATASE)(PHOSPHOTYROSINE PHOSPHATASE) (PTPASE). GENBANK ACCESSION NO: O43655PROTEIN TYROSINE PHOSPHATASE, RECEPTOR TYPE, R (EC 3.1.3.48) (RECEPTORPROTEIN TYROSINE PHOSPHATASE) (FRAGMENT). GENBANK ACCESSION NO: O75551PROTEIN PHOSPHATASE 2C ALPHA 2. GENBANK ACCESSION NO: Q64605 LEUKOCYTECOMMON ANTIGEN-RELATED PHOSPHATASE PTP2 PRECURSOR (EC 3.1.3.48)(PROTEIN-TYROSINE PHOSPHATASE LAR-PTP2) (PHOSPHOTYROSINE PHOSPHATASELAR-PTP2) (PTPASE LAR-PTP2) (PTP NE-3) (PTP-P1) (CPTP1) (PTP-SIGMA).GENBANK ACCESSION NO: PTPK_HUMAN PROTEIN-TYROSINE PHOSPHATASE KAPPAPRECURSOR (EC 3.1.3.48) (R-PTP-KAPPA). GENBANK ACCESSION NO: PP11_DROMESERINE/THREONINE PROTEIN PHOSPHATASE ALPHA-1 ISOFORM (EC 3.1.3.16).GENBANK ACCESSION NO: Q42981 SERINE/THREONINE PROTEIN PHOSPHATASE (EC3.1.3.16). GENBANK ACCESSION NO: O88740 PROTEIN-TYROSINE-PHOSPHATASE (EC3.1.3.48) (PHOSPHOTYROSINE PHOSPHATASE) (PTPASE). GENBANK ACCESSION NO:O81955 PP1A PROTEIN. GENBANK ACCESSION NO: PTNB MOUSE PROTEIN-TYROSINEPHOSPHATASE SYP (EC 3.1.3.48). GENBANK ACCESSION NO: O81956 PP2A1PROTEIN. GENBANK ACCESSION NO: P2BA_HUMAN SERINE/THREONINE PROTEINPHOSPHATASE 2B CATALYTIC SUBUNIT, ALPHA ISOFORM (EC 3.1.3.16)(CALMODULIN-DEPENDENT CALCINEURIN A SUBUNIT, ALPHA ISOFORM) (CAM-PRPCATALYTIC SUBUNIT). GENBANK ACCESSION NO: P2BA_BOVIN SERINE/THREONINEPROTEIN PHOSPHATASE 2B CATALYTIC SUBUNIT, ALPHA ISOFORM (EC 3.1.3.16)(CALMODULIN-DEPENDENT CALCINEURIN A SUBUNIT, ALPHA ISOFORM) (CAM-PRPCATALYTIC SUBUNIT). GENBANK ACCESSION NO: PT12_STYPL PROTEIN-TYROSINEPHOSPHATASE 12 (EC 3.1.3.48) (FRAGMENT). GENBANK ACCESSION NO:P2A4_ARATH SERINE/THREONINE PROTEIN PHOSPHATASE PP2A-4 CATALYTIC SUBUNIT(EC 3.1.3.16). GENBANK ACCESSION NO: PTPM_MOUSE PROTEIN-TYROSINEPHOSPHATASE MU PRECURSOR (EC 3.1.3.48) (R-PTP-MU). GENBANK ACCESSION NO:PCP2_HUMAN PROTEIN-TYROSINE PHOSPHATASE PCP-2 PRECURSOR (EC 3.1.3.48).GENBANK ACCESSION NO: P2BC_MOUSE SERINE/THREONINE PROTEIN PHOSPHATASE 2BCATALYTIC SUBUNIT, GAMMA ISOFORM (EC 3.1.3.16) (CALMODULIN-DEPENDENTCALCINEURIN A SUBUNIT, GAMMA ISOFORM) (CALCINEURIN, TESTIS-SPECIFICCATALYTIC SUBUNIT) (CAM- PRP CATALYTIC SUBUNIT). GENBANK ACCESSION NO:O00197 RECEPTOR PROTEIN TYROSINE PHOSPHATASE HPTP-J PRECURSOR (EC3.1.3.48). GENBANK ACCESSION NO: O61722 PUTATIVE PRENYLATED PROTEINTYROSINE PHOSPHATASE PRL-1. GENBANK ACCESSION NO: PPE1_SCHPOSERINE/THREONINE PROTEIN PHOSPHATASE PPE1 (EC 3.1.3.16) (PHOSPHATASEESP1). GENBANK ACCESSION NO: Q9XGT7 SERINE/THREONINE PROTEIN PHOSPHATASEPP2A-3 CATALYTIC SUBUNIT. GENBANK ACCESSION NO: PP14_ARATHSERINE/THREONINE PROTEIN PHOSPHATASE PP1 ISOZYME 4 (EC 3.1.3.16).GENBANK ACCESSION NO: O76451 SERINE/THREONINE PROTEIN PHOSPHATASE I(FRAGMENT). GENBANK ACCESSION NO: O35564 PROTEIN TYROSINE PHOSPHATASE,RECEPTOR TYPE, L (EC 3.1.3.48) (FTP-1). GENBANK ACCESSION NO: PPX1_ARATHSERINE/THREONINE PROTEIN PHOSPHATASE PP-X ISOZYME 1 (EC 3.1.3.16).GENBANK ACCESSION NO: O65844 PROTEIN PHOSPHATASE 1, CATALYTIC BETASUBUNIT. GENBANK ACCESSION NO: Q62917 LAR RECEPTOR-LINKED TYROSINEPHOSPHATASE (EC 3.1.3.48). GENBANK ACCESSION NO: O65845 PROTEINPHOSPHATASE 1, CATALYTIC GSMMS SUBUNIT. GENBANK ACCESSION NO: Q64538PHOSPHOPROTEIN PHOSPHATASE (FRAGMENT). GENBANK ACCESSION NO: O65846PROTEIN PHOSPHATASE 1 CATALITIC SUBUNIT. GENBANK ACCESSION NO:PTN3_HUMAN PROTEIN-TYROSINE PHOSPHATASE H1 (EC 3.1.3.48) (PTP-H1).GENBANK ACCESSION NO: P2C3_YEAST PROTEIN PHOSPHATASE 2C HOMOLOG 3 (EC3.1.3.16) (PP2C-3). GENBANK ACCESSION NO: O65847 PROTEIN PHOSPHATASE 1,CATALYTIC EPSILON SUBUNIT. GENBANK ACCESSION NO: PPP6_HUMANSERINE/THREONINE PROTEIN PHOSPHATASE 6 (EC 3.1.3.16) (PP6). GENBANKACCESSION NO: O88591 PROTEIN PHOSPHATASE TYPE 2A CATALYTIC SUBUNIT ALPHAISOFORM. GENBANK ACCESSION NO: P2AB_PIG SERINE/THREONINE PROTEINPHOSPHATASE PP2A-BETA, CATALYTIC SUBUNIT (EC 3.1.3.16) (FRAGMENT).GENBANK ACCESSION NO: PT06_STYPL PROTEIN-TYROSINE PHOSPHATASE 6 (EC3.1.3.48) (FRAGMENT). GENBANK ACCESSION NO: P2A_BRANA SERINE/THREONINEPROTEIN PHOSPHATASE PP2A CATALYTIC SUBUNIT (EC 3.1.3.16) (FRAGMENT).GENBANK ACCESSION NO: P2A_MEDSA SERINE/THREONINE PROTEIN PHOSPHATASEPP2A CATALYTIC SUBUNIT (EC 3.1.3.16). GENBANK ACCESSION NO: PTNC_HUMANPROTEIN-TYROSINE PHOSPHATASE G1 (EC 3.1.3.48) (PTPG1). GENBANK ACCESSIONNO: O15253 SERINE/THREONINE PROTEIN PHOSPHATASE. GENBANK ACCESSION NO:P2CB_MOUSE PROTEIN PHOSPHATASE 2C BETA ISOFORM (EC 3.1.3.16) (PP2C-BETA)(IA) (PROTEIN PHOSPHATASE 1B). GENBANK ACCESSION NO: Q61152PROTEIN-TYROSINE PHOSPHATASE 18 (EC 3.1.3.48) (PTP- K1) (FETAL LIVERPHOSPHATASE 1) (FLP1) (PTP 49) (PTP HSCF). GENBANK ACCESSION NO: O22626PROTEIN PHOSPHATASE X ISOFORM 2. GENBANK ACCESSION NO: Q9XGU3PHOSPHATASE PP1. GENBANK ACCESSION NO: PTPF_HUMAN LAR PROTEIN PRECURSOR(LEUKOCYTE ANTIGEN RELATED) (EC 3.1.3.48). GENBANK ACCESSION NO: Q64621RECEPTOR-LINKED PROTEIN TYROSINE PHOSPHATASE (EC 3.1.3.48)(PROTEIN-TYROSINE-PHOSPHATASE) (PHOSPHOTYROSINE PHOSPHATASE) (PTPASE).GENBANK ACCESSION NO: YME1_CAEEL PUTATIVE SERINE/THREONINE PROTEINPHOSPHATASE F56C9.1 IN CHROMOSOME III (EC 3.1.3.16). GENBANK ACCESSIONNO: Q64622 PROTEIN-TYROSINE-PHOSPHATASE (EC 3.1.3.48) (PHOSPHOTYROSINEPHOSPHATASE) (PTPASE) (FRAGMENT). GENBANK ACCESSION NO: Q24708PROTEIN-TYROSINE PHOSPHATASE CORKSCREW (EC 3.1.3.48) (CSW) (FRAGMENT).GENBANK ACCESSION NO: Q15718 PTPSIGMA PRECURSOR (EC 3.1.3.48). GENBANKACCESSION NO: PTPA_RAT PROTEIN-TYROSINE PHOSPHATASE ALPHA PRECURSOR (EC3.1.3.48) (R-PTP-ALPHA). GENBANK ACCESSION NO: Q63739 TYROSINEPHOSPHATASE. GENBANK ACCESSION NO: P91569 PROBABLE SERINE/THREONINEPROTEIN PHOSPHATASE (EC 3.1.3.16). GENBANK ACCESSION NO: WZB_ECOLIPROBABLE LOW MOLECULAR WEIGHT PROTEIN-TYROSINE- PHOSPHATASE WZB (EC3.1.3.48). GENBANK ACCESSION NO: PP1G_MOUSE SERINE/THREONINE PROTEINPHOSPHATASE PP1-GAMMA CATALYTIC SUBUNIT (EC 3.1.3.16) (PP-1G). GENBANKACCESSION NO: O88765 PROTEIN TYROSINE PHOSPHATASE. GENBANK ACCESSION NO:Q98945 PROTEIN TYROSINE PHOSPHATASE CRYP-2 PRECURSOR (EC 3.1.3.48).GENBANK ACCESSION NO: YOR5_KLEPN PUTATIVE LOW MOLECULAR WEIGHTPROTEIN-TYROSINE- PHOSPHATASE (EC 3.1.3.48) (ORF5). GENBANK ACCESSIONNO: P2BB_RAT SERINE/THREONINE PROTEIN PHOSPHATASE 2B CATALYTIC SUBUNIT,BETA ISOFORM (EC 3.1.3.16) (CALMODULIN-DEPENDENT CALCINEURIN A SUBUNIT,BETA ISOFORM) (CAM-PRP CATALYTIC SUBUNIT). GENBANK ACCESSION NO: Q9Y0B7PROTEIN PHOSPHATASE 4 CATALYTIC SUBUNIT. GENBANK ACCESSION NO: Q04071PROTEIN PHOSPHATASE 2B CATALYTIC SUBUNIT C (EC 3..3.16) (PP-2BC)(CALMODULIN-DEPENDENT CALCINEURIN A SUBUNIT) (FRAGMENT). GENBANKACCESSION NO: YWLE_BACSU PUTATIVE LOW MOLECULAR WEIGHT PROTEIN-TYROSINE-PHOSPHATASE (EC 3.1.3.48). GENBANK ACCESSION NO: Q9ZTF1 PUTATIVETRANSCRIPTION FACTOR (FRAGMENT). GENBANK ACCESSION NO: Q62132PROTEIN-TYROSINE PHOSPHATASE, RECEPTOR-TYPE, Q PRECURSOR (EC 3.1.3.48)(PROTEIN-TYROSINE-PHOSPHATASE SL) (PHOSPHOTYROSINE PHOSPHATASE). GENBANKACCESSION NO: P70602 PROTEIN TYROSINE PHOSPHATASE 20 (EC 3.1.3.48).GENBANK ACCESSION NO: P2A1_NEUCR SERINE/THREONINE PROTEIN PHOSPHATASEPP2A CATALYTIC SUBUNIT (EC 3.1.3.16). GENBANK ACCESSION NO: Q62135PROTEIN-TYROSINE PHOSPHATASE 13 (EC 3.1.3.48) (RIP). GENBANK ACCESSIONNO: O17047 PROTEIN PHOSPHATASE WITH EF-HANDS. GENBANK ACCESSION NO:O43049 SERINE/THREONINE PROTEIN PHOSPHATASE. GENBANK ACCESSION NO:Q9XF94 SERINE/THREONINE PROTEIN PHOSPHATASE PP2A-2 CATALYTIC SUBUNIT.GENBANK ACCESSION NO: PPP4_RABIT SERINE/THREONINE PROTEIN PHOSPHATASE 4(EC 3.1.3.16) (PP4) (PROTEIN PHOSPHATASE X) (PP-X). GENBANK ACCESSIONNO: PPZ_SCHPO SERINE/THREONINE PROTEIN PHOSPHATASE PP-Z (EC 3.1.3.16).GENBANK ACCESSION NO: Q12974 PROTEIN-TYROSINE PHOSPHATASE. GENBANKACCESSION NO: Q63745 PROTEIN TYROSINE PHOSPHATASE (EC 3.1.3.48). GENBANKACCESSION NO: P2A3_YEAST SERINE/THREONINE PROTEIN PHOSPHATASE PPH3 (EC3.1.3.16). GENBANK ACCESSION NO: P97470 SERINE/THREONINE PROTEINPHOSPHATASE (EC 3.1.3.16) (FRAGMENT). GENBANK ACCESSION NO: O75664DJ707K17.1 (RECEPTOR PROTEIN TYROSINE PHOSPHATASE (RPTP-RHO, EC3.1.3.48)) (EC 3.1.3.48) (FRAGMENT). GENBANK ACCESSION NO: Q62937 PP-1M(FRAGMENT). GENBANK ACCESSION NO: Q27786 SERINE/THREONINE PROTEINPHOSPHATASE (EC 3.1.3.16). GENBANK ACCESSION NO: Q27787 SERINE/THREONINEPROTEIN PHOSPHATASE (EC 3.1.3.16). GENBANK ACCESSION NO: PTN6_HUMANPROTEIN-TYROSINE PHOSPHATASE 1C (EC 3.1.3.48) (PTP-1C) (HEMATOPOIETICCELL PROTEIN-TYROSINE PHOSPHATASE) (SH-PTP1). GENBANK ACCESSION NO:Q60998 PROTEIN-TYROSINE PHOSPHATE PHI (EC 3.1.3.48) (PTP PHI). GENBANKACCESSION NO: PTPA_MYCTU PROBABLE LOW MOLECULAR WEIGHT PROTEIN-TYROSINE-PHOSPHATASE (EC 3.1.3.48) (PTPASE). GENBANK ACCESSION NO: PT09_STYPLPROTEIN-TYROSINE PHOSPHATASE 9 (EC 3.1.3.48) (FRAGMENT). GENBANKACCESSION NO: Q99849 PROTEIN TYROSINE PHOSPHATASE HOMOLOG HPRL-R(FRAGMENT). GENBANK ACCESSION NO: P2C LEICH PROTEIN PHOSPHATASE 2C (EC3.1.3.16) (PP2C). GENBANK ACCESSION NO: P2CA_RAT PROTEIN PHOSPHATASE 2CALPHA ISOFORM (EC 3.1.3.16) (PP2C-ALPHA) (IA) (PROTEIN PHOSPHATASE 1A).GENBANK ACCESSION NO: PTPA_HUMAN PROTEIN-TYROSINE PHOSPHATASE ALPHAPRECURSOR (EC 3.1.3.48) (R-PTP-ALPHA). GENBANK ACCESSION NO: P2A1_SCHPOMINOR SERINE/THREONINE PROTEIN PHOSPHATASE PP2A- 1 CATALYTIC SUBUNIT (EC3.1.3.16). GENBANK ACCESSION NO: PTN8_MOUSE HEMATOPOIETIC CELLPROTEIN-TYROSINE PHOSPHATASE 70Z-PEP (EC 3.1.3.48). GENBANK ACCESSIONNO: Q10728 SERINE/THREONINE PROTEIN PHOSPHATASE PP1 SMOOTH MUSCLEREGULATORY M110 SUBUNIT (110 KDA SUBUNIT). GENBANK ACCESSION NO: Q9YDZ2266AA LONG HYPOTHETICAL SERINE/THREONINE PROTEIN PHOSPHATASE PP2ACATALYTIC SUBUNIT. GENBANK ACCESSION NO: Q10729 SERINE/THREONINE PROTEINPHOSPHATASE PP1 SMOOTH MUSCLE REGULATORY M21 SUBUNIT (21 KDA SUBUNIT).GENBANK ACCESSION NO: PP1_BRANA SERINE/THREONINE PROTEIN PHOSPHATASE PP1(EC 3.1.3.16) (FRAGMENT). GENBANK ACCESSION NO: Q64641 BRAIN-ENRICHEDMEMBRANE-ASSOCIATED PROTEIN TYROSINE PHOSPHATASE (BEM)-1 (EC 3.1.3.48)(FRAGMENT). GENBANK ACCESSION NO: Q64642 BRAIN-ENRICHEDMEMBRANE-ASSOCIATED PROTEIN TYROSINE PHOSPHATASE 2 (EC 3.1.3.48) (BEM-2)(PROTEIN-TYROSINE-PHOSPHATASE) (PHOSPHOTYROSINE PHOSPHATASE) (PTPASE)(FRAGMENT). GENBANK ACCESSION NO: P2B2_YEAST SERINE/THREONINE PROTEINPHOSPHATASE 2B CATALYTIC SUBUNIT A2 (EC 3.1.3.16) (CALCINEURIN A2)(CALMODULIN-BINDING PROTEIN 2). GENBANK ACCESSION NO: O77294SERINE-THREONINE PROTEIN PHOSPHATASE. GENBANK ACCESSION NO: Q64486MPTPDELTA (EC 3.1.3.48) (PROTEIN-TYROSINE- PHOSPHATASE) (PHOSPHOTYROSINEPHOSPHATASE) (PTPASE) (FRAGMENT). GENBANK ACCESSION NO: PP11_SCHPOSERINE/THREONINE PROTEIN PHOSPHATASE PP1-1 (EC 3.1.3.16). GENBANKACCESSION NO: Q64487 PROTEIN-TYROSINE PHOSPHATASE, RECEPTOR-TYPE, DPRECURSOR (EC 3.1.3.48) (PROTEIN-TYROSINE PHOSPHATASE DELTA)(R-PTP-DELTA). GENBANK ACCESSION NO: PT10_STYPL PROTEIN-TYROSINEPHOSPHATASE 10 (EC 3.1.3.48) (FRAGMENT). GENBANK ACCESSION NO:EPSP_BURSO PROBABLE LOW MOLECULAR WEIGHT PROTEIN-TYROSINE- PHOSPHATASEEPSP (EC 3.1.3.48). GENBANK ACCESSION NO: P2A2_ARATH SERINE/THREONINEPROTEIN PHOSPHATASE PP2A-2 CATALYTIC SUBUNIT (EC 3.1.3.16). GENBANKACCESSION NO: PTPK_MOUSE PROTEIN-TYROSINE PHOSPHATASE KAPPA PRECURSOR(EC 3.1.3.48) (R-PTP-KAPPA). GENBANK ACCESSION NO: Q9XGH7 PROTEINPHOSPHATASE 2A CATALYTIC SUBUNIT (EC 3.1.3.16). GENBANK ACCESSION NO:Q00219 SERINE/THREONINE PROTEIN PHOSPHATASE PP1 (5.9) (EC 3.1.3.16).GENBANK ACCESSION NO: P2BA_MOUSE SERINE/THREONINE PROTEIN PHOSPHATASE 2BCATALYTIC SUBUNIT, ALPHA ISOFORM (EC 3.1.3.16) (CALMODULIN-DEPENDENTCALCINEURIN A SUBUNIT, ALPHA ISOFORM) (CAM-PRP CATALYTIC SUBUNIT).GENBANK ACCESSION NO: PP12_ARATH SERINE/THREONINE PROTEIN PHOSPHATASEPP1 ISOZYME 2 (EC 3.1.3.16). GENBANK ACCESSION NO: O43941 PROTEINPHOSPHATASE-2C. GENBANK ACCESSION NO: LAR_DROME PROTEIN-TYROSINEPHOSPHATASE DLAR PRECURSOR (EC 3.1.3.48) (PROTEIN-TYROSINE-PHOSPHATEPHOSPHOHYDROLASE). GENBANK ACCESSION NO: P2CA_RABIT PROTEIN PHOSPHATASE2C ALPHA ISOFORM (EC 3.1.3.16) (PP2C-ALPHA) (PROTEIN PHOSPHATASE 1A)(IA). GENBANK ACCESSION NO: Q07808 PROTEIN-TYROSINE PHOSPHATASE 1 (EC3.1.3.48) (PTPASE 1) (PTP-P1). GENBANK ACCESSION NO: Q90815PROTEIN-TYROSINE PHOSPHATASE (EC 3.1.3.48). GENBANK ACCESSION NO:P2A_DROME SERINE/THREONINE PROTEIN PHOSPHATASE PP2A (EC 3.1.3.16)(MICROTUBULE STAR PROTEIN). GENBANK ACCESSION NO: Q24495 RECEPTORPROTEIN-TYROSINE PHOSPHATASE PRECURSOR (EC 3.1.3.48). GENBANK ACCESSIONNO: Q90816 PROTEIN-TYROSINE PHOSPHATASE (FRAGMENT). GENBANK ACCESSIONNO: Q64653 PROTEIN TYROSINE PHOSPHATASE (EC 3.1.3.48) (PROTEIN-TYROSINE-PHOSPHATASE) (PHOSPHOTYROSINE PHOSPHATASE) (PTPASE) (FRAGMENT).GENBANK ACCESSION NO: Q63682 PROTEIN PHOSPHATASE-1A (FRAGMENT). GENBANKACCESSION NO: Y328_SYNY3 PUTATIVE LOW MOLECULAR WEIGHT PROTEIN-TYROSINE-PHOSPHATASE (EC 3.1.3.48). GENBANK ACCESSION NO: PPP4_HUMANSERINE/THREONINE PROTEIN PHOSPHATASE 4 (EC 3.1.3.16) (PP4) (PROTEINPHOSPHATASE X) (PP-X). GENBANK ACCESSION NO: YQF3_CAEEL PUTATIVESERINE/THREONINE PROTEIN PHOSPHATASE C34C12.3 IN CHROMOSOME III (EC3.1.3.16). GENBANK ACCESSION NO: Q64494 PROTEIN-TYROSINE PHOSPHATASE S(EC 3.1.3.48) (R-PTP- S) (FRAGMENT). GENBANK ACCESSION NO: Q64495PROTEIN-TYROSINE PHOSPHATASE DELTA (EC 3.1.3.48) (R- PTP-DELTA)(FRAGMENT). GENBANK ACCESSION NO: Q29585 PHOSPHOPROTEIN PHOSPHATASE (EC3.1.3.16) (SERINE/THREONINE SPECIFIC PROTEIN PHOSPHATASE) (PROTEINPHOSPHATASE-1) (PROTEIN PHOSPHATASE-2A) (PROTEIN PHOSPHATASE-2B)(PROTEIN PHOSPHATASE-2C) (FRAGMENT). GENBANK ACCESSION NO: Q64497PROTEIN-TYROSINE PHOSPHATASE BETA (EC 3.1.3.48) (R- PTP-BETA)(FRAGMENT). GENBANK ACCESSION NO: PP1_BRAOL SERINE/THREONINE PROTEINPHOSPHATASE PP1 (EC 3.1.3.16). GENBANK ACCESSION NO: Q62797 PROTEINTYROSINE PHOSPHATASE BK PRECURSOR (EC 3.1.3.48) (PTP-BK) (PROTEINTYROSINE PHOSPHATASE D30). GENBANK ACCESSION NO: O75688 PP2C PROTEIN.GENBANK ACCESSION NO: PT04_STYPL PROTEIN-TYROSINE PHOSPHATASE 4 (EC3.1.3.48) (FRAGMENT). GENBANK ACCESSION NO: Q13332 PROTEIN-TYROSINEPHOSPHATASE, RECEPTOR-TYPE, S PRECURSOR (EC 3.1.3.48) (PROTEIN-TYROSINEPHOSPHATASE SIGMA) (R-PTP-SIGMA) (PTPRS). GENBANK ACCESSION NO:YT91_CAEEL PUTATIVE SERINE/THREONINE PROTEIN PHOSPHATASE C06A1.3 INCHROMOSOME II (EC 3.1.3.16). GENBANK ACCESSION NO: PTPD_HUMANPROTEIN-TYROSINE PHOSPHATASE DELTA PRECURSOR (EC 3.1.3.48)(R-PTP-DELTA). GENBANK ACCESSION NO: O22662 PROTEIN PHOSPHATASE U(FRAGMENT). GENBANK ACCESSION NO: O15297 WIP1. GENBANK ACCESSION NO:PP12_DROME SERINE/THREONINE PROTEIN PHOSPHATASE ALPHA-2 ISOFORM (EC3.1.3.16). GENBANK ACCESSION NO: O62829 PROTEIN PHOSPHATASE 2C ALPHA (EC3.1.3.16). GENBANK ACCESSION NO: Q93095 PROTEIN TYROSINE PHOSPHATASE PEP(EC 3.1.3.48) (PROTEIN-TYROSINE-PHOSPHATASE) (PHOSPHOTYROSINEPHOSPHATASE) (PTPASE) (FRAGMENT). GENBANK ACCESSION NO: Q91556 PROTEINTYROSINE PHOSPHATASE ALPHA PRECURSOR (EC 3.1.3.48). GENBANK ACCESSIONNO: O52787 PTP PROTEIN. GENBANK ACCESSION NO: Q93096 PROTEIN TYROSINEPHOSPHATASE HPRL-1N (EC 3.1.3.48) (PROTEIN-TYROSINE-PHOSPHATASE)(PHOSPHOTYROSINE PHOSPHATASE) (PTPASE) (FRAGMENT). GENBANK ACCESSION NO:PTNC_MOUSE PROTEIN-TYROSINE PHOSPHATASE P19 (EC 3.1.3.48) (P19-PTP)(MPTP-PEST). GENBANK ACCESSION NO: Q62884 DENSITY-ENHANCED PHOSPHATASE-1PRECURSOR (EC 3.1.3.48) (DEP-1) (VASCULAR PROTEIN TYROSINE PHOSPHATASE1). GENBANK ACCESSION NO: P2BB_HUMAN SERINE/THREONINE PROTEINPHOSPHATASE 2B CATALYTIC SUBUNIT, BETA ISOFORM (EC 3.1.3.16)(CALMODULIN-DEPENDENT CALCINEURIN A SUBUNIT, BETA ISOFORM) (CAM-PRPCATALYTIC SUBUNIT). GENBANK ACCESSION NO: PPAC_BOVIN LOW MOLECULARWEIGHT PHOSPHOTYROSINE PROTEIN PHOSPHATASE (EC 3.1.3.48) (LOW MOLECULARWEIGHT CYTOSOLIC ACID PHOSPHATASE) (EC 3.1.3.2) (PTPASE). GENBANKACCESSION NO: Q99952 PROTEIN-TYROSINE-PHOSPHATASE (EC 3.1.3.48)(PHOSPHOTYROSINE PHOSPHATASE) (PTPASE). GENBANK ACCESSION NO: Q9YHE4PROTEIN TYROSINE PHOSPHATASE MEG1 (EC 3.1.3.48) (FRAGMENT). GENBANKACCESSION NO: Q9YHE5 PROTEIN TYROSINE PHOSPHATASE MEG1 (EC 3.1.3.48)(FRAGMENT). GENBANK ACCESSION NO: Q9YHE6 PROTEIN TYROSINE PHOSPHATASESH-PTP2 (EC 3.1.3.48) (FRAGMENT). GENBANK ACCESSION NO: Q9YHE7 PROTEINTYROSINE PHOSPHATASE H1 (EC 3.1.3.48) (FRAGMENT). GENBANK ACCESSION NO:O00810 PROTEIN TYROSINE PHOSPHATASE. GENBANK ACCESSION NO: PP1B_DROMESERINE/THREONINE PROTEIN PHOSPHATASE BETA ISOFORM (EC 3.1.3.16). GENBANKACCESSION NO: PPAC_RAT LOW MOLECULAR WEIGHT PHOSPHOTYROSINE PROTEINPHOSPHATASE ACP1/ACP2 (EC 3.1.3.48) (LOW MOLECULAR WEIGHT CYTOSOLIC ACIDPHOSPHATASE) (EC 3.1.3.2) (PTPASE). GENBANK ACCESSION NO: P2B1_DROMESERINE/THREONINE PROTEIN PHOSPHATASE 2B CATALYTIC SUBUNIT 1 (EC3.1.3.16) (CALMODULIN-DEPENDENT CALCINEURIN A1 SUBUNIT). GENBANKACCESSION NO: PPV_DROME SERINE/THREONINE PROTEIN PHOSPHATASE PP-V (EC3.1.3.16). GENBANK ACCESSION NO: Q24032 CORKSCREW PROTEIN Y1229 (EC3.1.3.48). GENBANK ACCESSION NO: Q24033 PROTEIN-TYROSINE PHOSPHATASECORKSCREW, ISOFORM 4A (EC 3.1.3.48) (CSW). GENBANK ACCESSION NO: Q42812SERINE/THREONINE PROTEIN PHOSPHATASE (EC 3.1.3.16). GENBANK ACCESSIONNO: O62830 PROTEIN PHOSPHATASE 2C BETA (EC 3.1.3.16). GENBANK ACCESSIONNO: Q95040 SERINE/THREONINE PROTEIN PHOSPHATASE (EC 3.1.3.16). GENBANKACCESSION NO: PP1_PHAVU SERINE/THREONINE PROTEIN PHOSPHATASE PP1 (EC3.1.3.16). GENBANK ACCESSION NO: P70643 RECEPTOR TYPE PROTEIN TYROSINEPHOPHATASE PSI (EC 3.1.3.48) (FRAGMENT). GENBANK ACCESSION NO:PP15_ARATH SERINE/THREONINE PROTEIN PHOSPHATASE PP1 ISOZYME 5 (EC3.1.3.16). GENBANK ACCESSION NO: P70644 RECEPTOR TYPE PROTEIN TYROSINEPHOSPHATASE MY (FRAGMENT). GENBANK ACCESSION NO: O18931 PROTEINPHOSPHATASE TYPE 1 BETA CATALYTIC SUBUNIT (FRAGMENT). GENBANK ACCESSIONNO: O04856 SERINE/THREONINE PROTEIN PHOSPHATASE (EC 3.1.3.16). GENBANKACCESSION NO: O18932 PROTEIN PHOSPHATASE 2A-ALPHA CATALYTIC SUBUNIT(FRAGMENT). GENBANK ACCESSION NO: O04857 SERINE/THREONINE PROTEINPHOSPHATASE (EC 3.1.3.16). GENBANK ACCESSION NO: O04858 SERINE/THREONINEPROTEIN PHOSPHATASE (EC 3.1.3.16). GENBANK ACCESSION NO: PPX2_ARATHSERINE/THREONINE PROTEIN PHOSPHATASE PP-X ISOZYME 2 (EC 3.1.3.16).GENBANK ACCESSION NO: P2A1_YEAST SERINE/THREONINE PROTEIN PHOSPHATASEPP2A-1 CATALYTIC SUBUNIT (EC 3.1.3.16). GENBANK ACCESSION NO: O04859SERINE/THREONINE PROTEIN PHOSPHATASE (EC 3.1.3.16). GENBANK ACCESSIONNO: Q9Y2R2 PROTEIN TYROSINE PHOSPHATASE HOMOLOG (EC 3.1.3.48). GENBANKACCESSION NO: Q9WU22 PROTEIN TYROSINE PHOSPHATASE MEG-01 (EC 3.1.3.48).GENBANK ACCESSION NO: O43966 PROTEIN PHOSPHATASE 2C. GENBANK ACCESSIONNO: PP1_MEDVA SERINE/THREONINE PROTEIN PHOSPHATASE PP1 (EC 3.1.3.16).GENBANK ACCESSION NO: Q64675 LEUKOCYTE COMMON ANTIGEN-RELATEDPHOSPHATASE PRECURSOR (EC 3.1.3.48) (PROTEIN-TYROSINE-PHOSPHATASE)(PHOSPHOTYROSINE PHOSPHATASE) (PTPASE). GENBANK ACCESSION NO: PTN4_HUMANPROTEIN-TYROSINE PHOSPHATASE MEG1 (EC 3.1.3.48) (PTPASE-MEG1) (MEG).GENBANK ACCESSION NO: P2CA_HUMAN PROTEIN PHOSPHATASE 2C ALPHA ISOFORM(EC 3.1.3.16) (PP2C-ALPHA) (IA) (PROTEIN PHOSPHATASE 1A). GENBANKACCESSION NO: P2AA_CHICK SERINE/THREONINE PROTEIN PHOSPHATASEPP2A-ALPHA, CATALYTIC SUBUNIT (EC 3.1.3.16). GENBANK ACCESSION NO:PT07_STYPL PROTEIN-TYROSINE PHOSPHATASE 7 (EC 3.1.3.48) (FRAGMENT).GENBANK ACCESSION NO: PP11_YEAST SERINE/THREONINE PROTEIN PHOSPHATASEPP1-1 (EC 3.1.3.16). GENBANK ACCESSION NO: PPAL_SCHPO LOW MOLECULARWEIGHT PHOSPHOTYROSINE PROTEIN PHOSPHATASE (EC 3.1.3.48) (LOW MOLECULARWEIGHT CYTOSOLIC ACID PHOSPHATASE) (EC 3.1.3.2) (PTPASE) (SMALL TYROSINEPHOSPHATASE). GENBANK ACCESSION NO: Q9Y879 CALCINEURIN A CATALYTICSUBUNIT. GENBANK ACCESSION NO: P2CB_RAT PROTEIN PHOSPHATASE 2C BETAISOFORM (EC 3.1.3.16) (PP2C-BETA) (IA) (PROTEIN PHOSPHATASE 1B). GENBANKACCESSION NO: O15712 PROTEIN PHOSPHATASE 2B. GENBANK ACCESSION NO:PPZ1_YEAST SERINE/THREONINE PROTEIN PHOSPHATASE PP-Z1 (EC 3.1.3.16).GENBANK ACCESSION NO: Q9X4B8 PUTATIVE ACID PHOSPHATASE WZB. GENBANKACCESSION NO: PTN6_MOUSE PROTEIN-TYROSINE PHOSPHATASE 1C (EC 3.1.3.48)(PTP-1C) (HEMATOPOIETIC CELL PROTEIN-TYROSINE PHOSPHATASE) (70Z-SHP)(SH-PTP1). GENBANK ACCESSION NO: O04860 SERINE/THREONINE PROTEINPHOSPHATASE (EC 3.1.3.16). GENBANK ACCESSION NO: P2C2_SCHPO PROTEINPHOSPHATASE 2C HOMOLOG 2 (EC 3.1.3.16) (PP2C-2). GENBANK ACCESSION NO:P2B1_CRYNE SERINE/THREONINE PROTEIN PHOSPHATASE 2B CATALYTIC SUBUNIT A1(EC 3.1.3.16) (CALCINEURIN A1). GENBANK ACCESSION NO: Q64046 MG2+DEPENDENT PROTEIN PHOSPHATASE BETA ISOFORM. GENBANK ACCESSION NO: Q61373PROTEIN TYROSINE PHOSPHATASE (EC 3.1.3.48) (FRAGMENT). GENBANK ACCESSIONNO: Q9XZE5 PROTEIN PHOSPHATASE 2A CATALYTIC SUBUNIT. GENBANK ACCESSIONNO: O81716 PROTEIN PHOSPHATASE 2C - LIKE PROTEIN. GENBANK ACCESSION NO:O14829 PROTEIN PHOSPHATASE WITH EF-HANDS-1. GENBANK ACCESSION NO: Q16826PROTEIN-TYROSINE-PHOSPHATASE (EC 3.1.3.48) (PHOSPHOTYROSINE PHOSPHATASE)(PTPASE). GENBANK ACCESSION NO: PP11_ACECL SERINE/THREONINE PROTEINPHOSPHATASE PP1 ISOZYME 1 (EC 3.1.3.16). GENBANK ACCESSION NO: Q16827PROTEIN-TYROSINE PHOSPHATASE, RECEPTOR-TYPE, O PRECURSOR (EC 3.1.3.48)(PROTEIN TYROSINE PHOSPHATASE U2) (GLOMERULAR EPITHELIAL PROTEIN 1)(GLEPP1) (PHOSPHOTYROSINE PHOSPHATASE U2) (PTPASE U2) (PTP-U2). GENBANKACCESSION NO: O75870 PTPSIGMA (EC 3.1.3.48) (FRAGMENT). GENBANKACCESSION NO: PTPA_MOUSE PROTEIN-TYROSINE PHOSPHATASE ALPHA PRECURSOR(EC 3.1.3.48) (R-PTP-ALPHA) (LCA-RELATED PHOSPHATASE). GENBANK ACCESSIONNO: O43979 SERINE-THREONINE PHOSPHOPROTEIN PHOSPHATASE. GENBANKACCESSION NO: O94748 PROTEIN PHOSPHATASE-Z-LIKE SERINE/THREONINE PROTEINPHOSPHATASE. GENBANK ACCESSION NO: Q90687 PROTEIN-TYROSINE PHOSPHATASEN11 (EC 3.1.3.48) (PROTEIN TYROSINE PHOSPHATASE, NON-RECEPTOR TYPE 11).GENBANK ACCESSION NO: PTP6_DROME PROTEIN-TYROSINE PHOSPHATASE DPTPPRECURSOR (EC 3.1.3.48) (PROTEIN-TYROSINE-PHOSPHATE PHOSPHOHYDROLASE).GENBANK ACCESSION NO: Q62987 PROTEIN TYROSINE PHOSPHATASE SH-PTP2(FRAGMENT). GENBANK ACCESSION NO: Q62988 PROTEIN TYROSINE PHOSPHATASEALPHA (FRAGMENT). GENBANK ACCESSION NO: Q62989 PROTEIN TYROSINEPHOSPHATASE GAMMA (FRAGMENT). GENBANK ACCESSION NO: AMSI_ERWAM PROBABLELOW MOLECULAR WEIGHT PROTEIN-TYROSINE- PHOSPHATASE AMSI (EC 3.1.3.48).GENBANK ACCESSION NO: PT16 STYPL PROTEIN-TYROSINE PHOSPHATASE 16 (EC3.1.3.48) (FRAGMENT). GENBANK ACCESSION NO: PPQ1_YEAST SERINE/THREONINEPROTEIN PHOSPHATASE PPQ (EC 3.1.3.16). GENBANK ACCESSION NO: PPY_DROMESERINE/THREONINE PROTEIN PHOSPHATASE PP-Y (EC 3.1.3.16). GENBANKACCESSION NO: O14830 PROTEIN PHOSPHATASE WITH EF-HANDS-2 LONG FORM.GENBANK ACCESSION NO: O14831 PROTEIN PHOSPHATASE WITH EF-HANDS-2 SHORTFORM. GENBANK ACCESSION NO: O04951 SERINE/THREONINE PROTEIN PHOSPHATASE(EC 3.1.3.16). GENBANK ACCESSION NO: O77023 DPP2C1. GENBANK ACCESSIONNO: Q42912 SERINE/THREONINE PROTEIN PHOSPHATASE (EC 3.1.3.16). GENBANKACCESSION NO: RDGC_DROME SERINE/THREONINE PROTEIN PHOSPHATASE RDGC (EC3.1.3.16) (RETINAL DEGENERATION C PROTEIN). GENBANK ACCESSION NO: O76932SERINE/THREONINE SPECIFIC PROTEIN PHOSPHATASE 4 (EC 3.1.3.16). GENBANKACCESSION NO: P2AA_HUMAN SERINE/THREONINE PROTEIN PHOSPHATASEPP2A-ALPHA, CATALYTIC SUBUNIT (EC 3.1.3.16) (REPLICATION PROTEIN C)(RP-C). GENBANK ACCESSION NO: P2A4_YEAST SERINE/THREONINE PROTEINPHOSPHATASE PP2A-LIKE PPG1 (EC 3.1.3.16). GENBANK ACCESSION NO: Q9W6R4PROTEIN PHOSPHATASE 1. GENBANK ACCESSION NO: PP1_EMENI SERINE/THREONINEPROTEIN PHOSPHATASE PP1 (EC 3.1.3.16). GENBANK ACCESSION NO: O59927SERINE/THREONINE PROTEIN PHOSPHATASE TYPE 1. GENBANK ACCESSION NO:PTPA_STRCO LOW MOLECULAR WEIGHT PROTEIN-TYROSINE- PHOSPHATASE (EC3.1.3.48) (PTPASE) (SMALL, ACIDIC PHOSPHOTYROSINE PROTEIN PHOSPHATASE)(PY PROTEIN PHOSPHATASE). GENBANK ACCESSION NO: Q64696 PROTEIN-TYROSINEPHOSPHATASE, RECEPTOR-TYPE, F POLYPEPTIDE (EC 3.1.3.48) (LAR PROTEIN)(LEUKOCYTE ANTIGEN RELATED) (FRAGMENT). GENBANK ACCESSION NO: PTN7_HUMANPROTEIN-TYROSINE PHOSPHATASE LC-PTP (EC 3.1.3.48) (HEMATOPOIETICPROTEIN-TYROSINE PHOSPHATASE) (HEPTP). GENBANK ACCESSION NO: CSW_DROMEPROTEIN-TYROSINE PHOSPHATASE CORKSCREW (EC 3.1.3.48). GENBANK ACCESSIONNO: Q64699 PROTEIN-TYROSINE PHOSPHATASE, RECEPTOR-TYPE, S PRECURSOR (EC3.1.3.48) (PROTEIN-TYROSINE PHOSPHATASE SIGMA) (RPTP-SIGMA) (PROTEINTYROSINE PHOSPHATASE PTPT9) (PTPASE NU-3). GENBANK ACCESSION NO:PP11_TRYBB SERINE/THREONINE PROTEIN PHOSPHATASE PP1(4.8) (EC 3.1.3.16).GENBANK ACCESSION NO: PP1_MAIZE SERINE/THREONINE PROTEIN PHOSPHATASE PP1(EC 3.1.3.16). GENBANK ACCESSION NO: PT25_STYPL PROTEIN-TYROSINEPHOSPHATASE 25 (EC 3.1.3.48) (FRAGMENT). GENBANK ACCESSION NO:PP1A_HUMAN SERINE/THREONINE PROTEIN PHOSPHATASE PP1-ALPHA 1 CATALYTICSUBUNIT (EC 3.1.3.16) (PP-1A). GENBANK ACCESSION NO: PPP5_RATSERINE/THREONINE PROTEIN PHOSPHATASE 5 (EC 3.1.3.16) (PP5) (PROTEINPHOSPHATASE T) (PPT). GENBANK ACCESSION NO: PTPB_HUMAN PROTEIN-TYROSINEPHOSPHATASE BETA PRECURSOR (EC 3.1.3.48) (R-PTP-BETA). GENBANK ACCESSIONNO: P2C1_CAEEL PROBABLE PROTEIN PHOSPHATASE 2C F42G9.1 (EC 3.1.3.16)(PP2C). GENBANK ACCESSION NO: P2A2_SCHPO MAJOR SERINE/THREONINE PROTEINPHOSPHATASE PP2A- 2 CATALYTIC SUBUNIT (EC 3.1.3.16). GENBANK ACCESSIONNO: O44328 RECEPTOR TYROSINE PHOSPHATASE (EC 3.1.3.48). GENBANKACCESSION NO: O94044 PHOSPHOTYROSINE PROTEIN PHOSPHATASE. GENBANKACCESSION NO: O44329 RECEPTOR TYROSINE PHOSPHATASE (EC 3.1.3.48).GENBANK ACCESSION NO: PTPJ_HUMAN PROTEIN-TYROSINE PHOSPHATASE ETAPRECURSOR (EC 3.1.3.48) (R-PTP-ETA) (DENSITY ENHANCED PHOSPHATASE-1)(DEP-1) (CD148 ANTIGEN). GENBANK ACCESSION NO: Q9YI74 SERINE/THREONINEPHOSPHATASE. GENBANK ACCESSION NO: O08367 SERINE/THREONINE SPECIFICPROTEIN PHOSPHATASE (EC 3.1.3.16) (SERINE/THREONINE SPECIFIC PROTEINPHOSPHATASE) (PHOSPHOPROTEIN PHOSPHATASE) (PROTEIN PHOSPHATASE-1)(PROTEIN PHOSPHATASE-2A) (PROTEIN PHOSPHATASE-2B) (PROTEINPHOSPHATASE-2C). GENBANK ACCESSION NO: Q9YI75 SERINE/THREONINEPHOSPHATASE. GENBANK ACCESSION NO: Q9YI76 SERINE/THREONINE PHOSPHATASE.GENBANK ACCESSION NO: O57438 CALCINEURIN A. GENBANK ACCESSION NO:PTP1_DROME PROTEIN-TYROSINE PHOSPHATASE 10D PRECURSOR (EC 3.1.3.48)(RECEPTOR-LINKED PROTEIN-TYROSINE PHOSPHATASE 10D). GENBANK ACCESSIONNO: O82469 PROTEIN PHOSPHATASE-2C. GENBANK ACCESSION NO: PP12_SCHPOSERINE/THREONINE PROTEIN PHOSPHATASE PP1-2 (EC 3.1.3.16) (SUPPRESSORPROTEIN SDS21). GENBANK ACCESSION NO: PT11_STYPL PROTEIN-TYROSINEPHOSPHATASE 11 (EC 3.1.3.48) (FRAGMENT). GENBANK ACCESSION NO:PTP9_DROME PROTEIN-TYROSINE PHOSPHATASE 99A PRECURSOR (EC 3.1.3.48)(RECEPTOR-LINKED PROTEIN-TYROSINE PHOSPHATASE 99A). GENBANK ACCESSIONNO: Q9Y1W9 SPTPN6 (EC 3.1.3.48) (FRAGMENT). GENBANK ACCESSION NO:P2A3_ARATH SERINE/THREONINE PROTEIN PHOSPHATASE PP2A-3 CATALYTIC SUBUNIT(EC 3.1.3.16). GENBANK ACCESSION NO: PTPO_RAT OSTEOTESTICULAR PROTEINTYROSINE PHOSPHATASE PRECURSOR (EC 3.1.3.48) (OST-PTP). GENBANKACCESSION NO: P2BB_MOUSE SERINE/THREONINE PROTEIN PHOSPHATASE 2BCATALYTIC SUBUNIT, BETA ISOFORM (EC 3.1.3.16) (CALMODULIN-DEPENDENTCALCINEURIN A SUBUNIT, BETA ISOFORM) (CAM-PRP CATALYTIC SUBUNIT)(FRAGMENT). GENBANK ACCESSION NO: Q14513 TYROSINE PHOSPHATASE PRECURSOR(EC 3.1.3.48). GENBANK ACCESSION NO: PP1G_XENLA SERINE/THREONINE PROTEINPHOSPHATASE PP1-GAMMA CATALYTIC SUBUNIT (EC 3.1.3.16) (PP-1G). GENBANKACCESSION NO: P70125 PROTEIN TYROSINE PHOSPHATASE, RECEPTOR TYPE, L (EC3.1.3.48) (RECEPTOR PROTEIN TYROSINE PHOSPHATASE-LAMDA). GENBANKACCESSION NO: P2B1_SCHPO SERINE/THREONINE PROTEIN PHOSPHATASE 2BCATALYTIC SUBUNIT (EC 3.1.3.16). GENBANK ACCESSION NO: Q23345 SIMILAR TOOTHER PROTEIN PHOSPHATASES 1. GENBANK ACCESSION NO: PPAL_YEAST LOWMOLECULAR WEIGHT PHOSPHOTYROSINE PROTEIN PHOSPHATASE (EC 3.1.3.48) (LOWMOLECULAR WEIGHT CYTOSOLIC ACID PHOSPHATASE) (EC 3.1.3.2) (PTPASE).GENBANK ACCESSION NO: PP13_ARATH SERINE/THREONINE PROTEIN PHOSPHATASEPP1 ISOZYME 3 (EC 3.1.3.16). GENBANK ACCESSION NO: O82470 PROTEINPHOSPHATASE-2C. GENBANK ACCESSION NO: O82471 PROTEIN PHOSPHATASE-2C.GENBANK ACCESSION NO: O49346 A SERINE/THREONINE PROTEIN PHOSPHATASE (EC3.1.3.16) (SERINE/THREONINE SPECIFIC PROTEIN PHOSPHATASE)(PHOSPHOPROTEIN PHOSPHATASE) (PROTEIN PHOSPHATASE-1) (PROTEINPHOSPHATASE-2A) (PROTEIN PHOSPHATASE-2B) (PROTEIN PHOSPHATASE-2C).GENBANK ACCESSION NO: YSD1_CAEEL PUTATIVE SERINE/THREONINE PROTEINPHOSPHATASE C23G10.1 IN CHROMOSOME II (EC 3.1.3.16). GENBANK ACCESSIONNO: PTN2_HUMAN T-CELL PROTEIN-TYROSINE PHOSPHATASE (EC 3.1.3.48)(TCPTP). GENBANK ACCESSION NO: P2C2_YEAST PROTEIN PHOSPHATASE 2C HOMOLOG2 (EC 3.1.3.16) (PP2C-2). GENBANK ACCESSION NO: PPP5_HUMANSERINE/THREONINE PROTEIN PHOSPHATASE 5 (EC 3.1.3.16) (PP5) (PROTEINPHOSPHATASE T) (PP-T) (PPT). GENBANK ACCESSION NO: O82479 PROTEINPHOSPHATASE-2C (FRAGMENT). GENBANK ACCESSION NO: P2C PARTE PROTEINPHOSPHATASE 2C (EC 3.1.3.16) (PP2C). GENBANK ACCESSION NO: Q9Y1X5SPTPR2B (EC 3.1.3.48) (FRAGMENT). GENBANK ACCESSION NO: Q9Y1X6 SPTPR4(EC 3.1.3.48) (FRAGMENT). GENBANK ACCESSION NO: P2CG_HUMAN PROTEINPHOSPHATASE 2C GAMMA ISOFORM (EC 3.1.3.16) (PP2C-GAMMA) (PROTEINPHOSPHATASE 1C). GENBANK ACCESSION NO: P2CG_BOVIN PROTEIN PHOSPHATASE 2CGAMMA ISOFORM (EC 3.1.3.16) (PP2C-GAMMA) (PROTEIN PHOSPHATASE 1B)(MAGNESIUM-DEPENDENT CALCIUM INHIBITABLE PHOSPHATASE) (MCPP). GENBANKACCESSION NO: P2A_HELAN SERINE/THREONINE PROTEIN PHOSPHATASE PP2ACATALYTIC SUBUNIT (EC 3.1.3.16). GENBANK ACCESSION NO: PTNB_HUMANPROTEIN-TYROSINE PHOSPHATASE 2C (EC 3.1.3.48) (PTP-2C) (PTP-1D)(SH-PTP3) (SH-PTP2) (SHP-2). GENBANK ACCESSION NO: P2CA_MOUSE PROTEINPHOSPHATASE 2C ALPHA ISOFORM (EC 3.1.3.16) (PP2C-ALPHA) (IA) (PROTEINPHOSPHATASE 1A). GENBANK ACCESSION NO: P91420 PROBABLE SERINE/THREONINEPROTEIN PHOSPHATASE (EC 3.1.3.16). GENBANK ACCESSION NO: PTPE_HUMANPROTEIN-TYROSINE PHOSPHATASE EPSILON PRECURSOR (EC 3.1.3.48)(R-PTP-EPSILON). GENBANK ACCESSION NO: Q15255 PROTEIN-TYROSINEPHOSPHATASE ETA PRECURSOR (EC 3.1.3.48) (R-PTP-ETA). GENBANK ACCESSIONNO: Q91054 CD45 HOMOLOG (EC 3.1.3.48). GENBANK ACCESSION NO: Q15256PROTEIN-TYROSINE PHOSPHATASE PCPTP1 PRECURSOR (EC 3.1.3.48)(PROTEIN-TYROSINE-PHOSPHATASE PCPTP1) (NC-PTPCOM1). GENBANK ACCESSIONNO: SD22_SCHPO PROTEIN PHOSPHATASES PP1 REGULATORY SUBUNIT SDS22.GENBANK ACCESSION NO: O15757 PROTEIN PHOSPHATASE TYPE 1-LIKE CATALYTICSUBUNIT. GENBANK ACCESSION NO: PTPM_HUMAN PROTEIN-TYROSINE PHOSPHATASEMU PRECURSOR (EC 3.1.3.48) (R-PTP-MU). GENBANK ACCESSION NO: PP13_DROMESERINE/THREONINE PROTEIN PHOSPHATASE ALPHA-3 ISOFORM (EC 3.1.3.16).GENBANK ACCESSION NO: Q27475 PROBABLE SERINE/THREONINE PROTEINPHOSPHATASE (EC 3.1.3.16). GENBANK ACCESSION NO: PTNB_RATPROTEIN-TYROSINE PHOSPHATASE SYP (EC 3.1.3.48). GENBANK ACCESSION NO:P2BC_HUMAN SERINE/THREONINE PROTEIN PHOSPHATASE 2B CATALYTIC SUBUNIT,GAMMA ISOFORM (EC 3.1.3.16) (CALMODULIN-DEPENDENT CALCINEURIN A SUBUNIT,GAMMA ISOFORM) (CALCINEURIN, TESTIS-SPECIFIC CATALYTIC SUBUNIT) (CAM-PRP CATALYTIC SUBUNIT). GENBANK ACCESSION NO: Q95097 SERINE/THREONINEPROTEIN PHOSPHATASE (EC 3.1.3.16). GENBANK ACCESSION NO: Q9ZSQ7 PROTEINPHOSPHATASE 2C HOMOLOG. GENBANK ACCESSION NO: YD44_SCHPO PUTATIVESERINE/THREONINE PROTEIN PHOSPHATASE C22H10.04 (EC 3.1.3.16). GENBANKACCESSION NO: Q9WUV7 SERINE/THREONINE SPECIFIC PROTEIN PHOSPHATASE (EC3.1.3.16). GENBANK ACCESSION NO: PPX1_PARTE SERINE/THREONINE PROTEINPHOSPHATASE PP-X HOMOLOG (EC 3.1.3.16). GENBANK ACCESSION NO: PTPO_MOUSEEMBRYONIC STEM CELL PROTEIN TYROSINE PHOSPHATASE PRECURSOR (EC 3.1.3.48)(ES CELL PHOSPHATASE). GENBANK ACCESSION NO: P2B2_DROME SERINE/THREONINEPROTEIN PHOSPHATASE 2B CATALYTIC SUBUNIT 2, (EC 3.1.3.16)(CALMODULIN-DEPENDENT CALCINEURIN A2 SUBUNIT). GENBANK ACCESSION NO:Q9Z1G2 SERINE/THREONINE PROTEIN PHOSPHATASE TYPE 1 ALPHA. GENBANKACCESSION NO: Q07161 PROTEIN PHOSPHATASE PP1-ALPHA 2, CATALYTIC SUBUNIT(EC 3.1.3.16). GENBANK ACCESSION NO: O15920 PROTEIN PHOSPHATASE-BETA.GENBANK ACCESSION NO: P2B_EMENI SERINE/THREONINE PROTEIN PHOSPHATASE 2BCATALYTIC SUBUNIT (EC 3.1.3.16) (CALMODULIN-DEPENDENT CALCINEURIN ASUBUNIT). GENBANK ACCESSION NO: YY06_CAEEL PUTATIVE SERINE/THREONINEPROTEIN PHOSPHATASE C27B7.6 IN CHROMOSOME IV (EC 3.1.3.16). GENBANKACCESSION NO: Q29500 PROTEIN-TYROSINE-PHOSPHATASE (EC 3.1.3.48)(PHOSPHOTYROSINE PHOSPHATASE) (PTPASE). GENBANK ACCESSION NO: Q15263PROTEIN TYROSINE PHOSPHATASE (PTP-BAS, TYPE 1). GENBANK ACCESSION NO:Q15264 PROTEIN TYROSINE PHOSPHATASE (PTP-BAS, TYPE 2). GENBANK ACCESSIONNO: Q15426 PROTEIN-TYROSINE PHOSPHATASE, RECEPTOR-TYPE, H PRECURSOR (EC3.1.3.48) (PROTEIN TYROSINE PHOSPHATASE SAP-1) (STOMACH CANCER-ASSOCIATED PTP). GENBANK ACCESSION NO: PP12_RABIT SERINE/THREONINEPROTEIN PHOSPHATASE PP1-ALPHA 2 CATALYTIC SUBUNIT (EC 3.1.3.16) (PP-1A).GENBANK ACCESSION NO: O02658 SERINE/THREONINE PROTEIN PHOSPHATASE (EC3.1.3.16). GENBANK ACCESSION NO: Q15265 PROTEIN TYROSINE PHOSPHATASE(PTP-BAS, TYPE 3). GENBANK ACCESSION NO: O70275 PROTEIN TYROSINEPHOSPHATASE 4A3 (MPRL-3). GENBANK ACCESSION NO: Q27560 SERINE/THREONINEPROTEIN PHOSPHATASE CALCINEURIN A (EC 3.1.3.16). GENBANK ACCESSION NO:O82733 SERINE/THREONINE PROTEIN PHOSPHATASE TYPE ONE. GENBANK ACCESSIONNO: P2AB_RABIT SERINE/THREONINE PROTEIN PHOSPHATASE PP2A-BETA, CATALYTICSUBUNIT (EC 3.1.3.16). GENBANK ACCESSION NO: PP16_ARATH SERINE/THREONINEPROTEIN PHOSPHATASE PP1 ISOZYME 6 (EC 3.1.3.16). GENBANK ACCESSION NO:P91273 PROBABLE SERINE/THREONINE PROTEIN PHOSPHATASE (EC 3.1.3.16).GENBANK ACCESSION NO: O82734 SERINE/THREONINE PROTEIN PHOSPHATASE TYPEONE. GENBANK ACCESSION NO: O75365 HPRL-3. GENBANK ACCESSION NO: P81718PROTEIN-TYROSINE PHOSPHATASE N6 (EC 3.1.3.48). GENBANK ACCESSION NO:P2A_ACECL SERINE/THREONINE PROTEIN PHOSPHATASE PP2A-1 CATALYTIC SUBUNIT(EC 3.1.3.16). GENBANK ACCESSION NO: P2A2_YEAST SERINE/THREONINE PROTEINPHOSPHATASE PP2A-2 CATALYTIC SUBUNIT (EC 3.1.3.16). GENBANK ACCESSIONNO: Q9W6V5 SUPPORTING-CELL ANTIGEN PRECURSOR (EC 3.1.3.48). GENBANKACCESSION NO: Q04101 PROTEIN PHOSPHATASE PP1-BETA CATALYTIC SUBUNIT (EC3.1.3.16) (FRAGMENT). GENBANK ACCESSION NO: PP12 YEAST SERINE/THREONINEPROTEIN PHOSPHATASE PP1-2 (EC 3.1.3.16). GENBANK ACCESSION NO: Q04102PROTEIN PHOSPHATASE PP1-C CATALYTIC SUBUNIT (EC 3.1.3.16) (FRAGMENT).GENBANK ACCESSION NO: Q04103 PROTEIN PHOSPHATASE PP1-D CATALYTIC SUBUNIT(EC 3.1.3.16) (FRAGMENT). GENBANK ACCESSION NO: PPT1_YEASTSERINE/THREONINE PROTEIN PHOSPHATASE T (EC 3.1.3.16) (PPT). GENBANKACCESSION NO: Q04104 PROTEIN PHOSPHATASE PP-X CATALYTIC SUBUNIT (EC3.1.3.16) (FRAGMENT). GENBANK ACCESSION NO: P2AA_MOUSE SERINE/THREONINEPROTEIN PHOSPHATASE PP2A-ALPHA, CATALYTIC SUBUNIT (EC 3.1.3.16). GENBANKACCESSION NO: PPP6_RAT SERINE/THREONINE PROTEIN PHOSPHATASE 6 (EC3.1.3.16) (PP6) (PROTEIN PHOSPHATASE V) (PP-V). GENBANK ACCESSION NO:PP1G_HUMAN SERINE/THREONINE PROTEIN PHOSPHATASE PP1-GAMMA CATALYTICSUBUNIT (EC 3.1.3.16) (PP-1G). GENBANK ACCESSION NO: PPZ2_YEASTSERINE/THREONINE PROTEIN PHOSPHATASE PP-Z2 (EC 3.1.3.16). GENBANKACCESSION NO: Q64501 PROTEIN TYROSINE PHOSPHATASE D28 (EC 3.1.3.48)(PROTEIN-TYROSINE-PHOSPHATASE) (PHOSPHOTYROSINE PHOSPHATASE) (PTPASE)(FRAGMENT). GENBANK ACCESSION NO: Q64502 PROTEIN TYROSINE PHOSPHATASE(EC 3.1.3.48) (PROTEIN- TYROSINE-PHOSPHATASE) (PHOSPHOTYROSINEPHOSPHATASE) (PTPASE) (FRAGMENT). GENBANK ACCESSION NO: Q12923PROTEIN-TYROSINE PHOSPHATASE, NONRECEPTOR-TYPE, 13 (EC 3.1.3.48)(PROTEIN-TYROSINE PHOSPHATASE 1E) (PTP-BAS, TYPE 1) (PROTEIN- TYROSINEPHOSPHATASE PTPL1) (PROTEIN-TYROSINE PHOSPHATASE 1, FAS-ASSOCIATED)(FAP-1). GENBANK ACCESSION NO: Q92124 PHOSPHOTYROSYL-PROTEIN PHOSPHATASE(EC 3.1.3.48) (PROTEIN-TYROSINE-PHOSPHATASE) (PHOSPHOTYROSINEPHOSPHATASE) (PTPASE). GENBANK ACCESSION NO: Q64503 PROTEIN TYROSINEPHOSPHATASE, RECEPTOR TYPE, S PRECURSOR (EC 3.1.3.48) (PHOSPHOTYROSINEPHOSPHATASE) (PTPASE). GENBANK ACCESSION NO: Q64504PROTEIN-TYROSINE-PHOSPHATASE (EC 3.1.3.48) (PHOSPHOTYROSINE PHOSPHATASE)(PTPASE) (FRAGMENT). GENBANK ACCESSION NO: P2C3_SCHPO PROTEINPHOSPHATASE 2C HOMOLOG 3 (EC 3.1.3.16) (PP2C-3). GENBANK ACCESSION NO:O48641 PROTEIN PHOSPHATASE 1 CATALYTIC SUBUNIT. GENBANK ACCESSION NO:Q15197 PROTEIN TYROSINE PHOSPHATASE (FRAGMENT). GENBANK ACCESSION NO:Q27573 SERINE/THREONINE PROTEIN PHOSPHATASE (EC 3.1.3.16). GENBANKACCESSION NO: Q63294 LEUKOCYTE COMMON ANTIGEN RELATED PROTEIN (EC3.1.3.48) (FRAGMENT). GENBANK ACCESSION NO: Q64509 PROTEIN TYROSINEPHOSPHATASE, NON-RECEPTOR TYPE 11 (EC 3.1.3.48)(PROTEIN-TYROSINE-PHOSPHATASE) (PHOSPHOTYROSINE PHOSPHATASE) (PTPASE).GENBANK ACCESSION NO: PP12_ACECL SERINE/THREONINE PROTEIN PHOSPHATASEPP1 ISOZYME 2 (EC 3.1.3.16). GENBANK ACCESSION NO: P2B1_YEASTSERINE/THREONINE PROTEIN PHOSPHATASE 2B CATALYTIC SUBUNIT A1 (EC3.1.3.16) (CALCINEURIN A1) (CALMODULIN-BINDING PROTEIN 1). GENBANKACCESSION NO: Q63295 LEUCOCYTE COMMON ANTIGEN-RELATED PROTEIN (EC3.1.3.48) (LAR) (FRAGMENT). GENBANK ACCESSION NO: O35299 PROTEINPHOSPHATASE 5. GENBANK ACCESSION NO: Q63296 LEUCOCYTE COMMONANTIGEN-RELATED PROTEIN (EC 3.1.3.48) (FRAGMENT). GENBANK ACCESSION NO:Q92682 PROTEIN-TYROSINE PHOSPHATASE NC-PTPCOM1 (EC 3.1.3.48)(PROTEIN-TYROSINE-PHOSPHATASE). GENBANK ACCESSION NO: Q9ZSS3 PROTEINPHOSPHATASE 2A CATALYTIC SUBUNIT. GENBANK ACCESSION NO: PP1_ORYSASERINE/THREONINE PROTEIN PHOSPHATASE PP1 (EC 3.1.3.16). GENBANKACCESSION NO: P2B_NEUCR SERINE/THREONINE PROTEIN PHOSPHATASE 2BCATALYTIC SUBUNIT (EC 3.1.3.16) (CALMODULIN-DEPENDENT CALCINEURIN ASUBUNIT). GENBANK ACCESSION NO: P2A1_ARATH SERINE/THREONINE PROTEINPHOSPHATASE PP2A-1 CATALYTIC SUBUNIT (EC 3.1.3.16). GENBANK ACCESSIONNO: YCCY_ECOLI PROBABLE LOW MOLECULAR WEIGHT PROTEIN-TYROSINE-PHOSPHATASE YCCY (EC 3.1.3.48). GENBANK ACCESSION NO: P78399 PROTEINTYROSINE PHOSPHATASE RECEPTOR OMICRON (EC 3.1.3.48). GENBANK ACCESSIONNO: PTPJ_MOUSE PROTEIN-TYROSINE PHOSPHATASE ETA PRECURSOR (EC 3.1.3.48)(R-PTP-ETA) (HPTP BETA-LIKE TYROSINE PHOSPHATASE). GENBANK ACCESSION NO:PT17_STYPL PROTEIN-TYROSINE PHOSPHATASE 17 (EC 3.1.3.48) (FRAGMENT).GENBANK ACCESSION NO: PP11_ARATH SERINE/THREONINE PROTEIN PHOSPHATASEPP1 ISOZYME 1 (EC 3.1.3.16). GENBANK ACCESSION NO: Q9ZRF6SERINE/THREONINE PROTEIN PHOSPHATASE 2A-3 CATALYTIC SUBUNIT. GENBANKACCESSION NO: Q64512 PROTEIN-TYROSINE PHOSPHATASE, NONRECEPTOR-TYPE, 13(EC 3.1.3.48) (PROTEIN-TYROSINE PHOSPHATASE RIP) (PHOSPHOPROTEINPHOSPHATASE) (PROTEIN-TYROSINE-PHOSPHATASE) (PHOSPHOTYROSINEPHOSPHATASE) (PTPASE) (PTP36). GENBANK ACCESSION NO: O75702PROTEIN-TYROSINE-PHOSPHATASE, ISOFORM 3 (EC 3.1.3.48) (PHOSPHOTYROSINEPHOSPHATASE) (PTPASE). GENBANK ACCESSION NO: O95063 LYMPHOID PHOSPHATASELYP1 (EC 3.1.3.48). GENBANK ACCESSION NO: O95064 LYMPHOID PHOSPHATASELYP2 (EC 3.1.3.48). GENBANK ACCESSION NO: O35385 PROTEIN PHOSPHATASEWITH EF-HANDS-2. GENBANK ACCESSION NO: Q92850 RECEPTOR PROTEIN TYROSINEPHOSPHATASE PSI (EC 3.1.3.48). GENBANK ACCESSION NO: O96914 PROTEINSERINE/THREONINE PHOSPHATASE ALPHA. GENBANK ACCESSION NO: P2AB_HUMANSERINE/THREONINE PROTEIN PHOSPHATASE PP2A-BETA, CATALYTIC SUBUNIT (EC3.1.3.16). GENBANK ACCESSION NO: P2A_PARTE SERINE/THREONINE PROTEINPHOSPHATASE PP2A CATALYTIC SUBUNIT (EC 3.1.3.16) (PPN). GENBANKACCESSION NO: O88739 PROTEIN-TYROSINE-PHOSPHATASE (EC 3.1.3.48)(PHOSPHOTYROSINE PHOSPHATASE) (PTPASE). GENBANK ACCESSION NO: Q91969PROTEIN TYROSINE PHOSPHATASE PRECURSOR (EC 3.1.3.48). GENBANK ACCESSIONNO: PP12_TRYBB SERINE/THREONINE PROTEIN PHOSPHATASE PP1 (5.9) (EC3.1.3.16). GENBANK ACCESSION NO: PP1B_HUMAN SERINE/THREONINE PROTEINPHOSPHATASE PP1-BETA CATALYTIC SUBUNIT (EC 3.1.3.16) (PP-1B). GENBANKACCESSION NO: O42205 PROTEIN PHOSPHATASE 5 (FRAGMENT). GENBANK ACCESSIONNO: PPP5_MOUSE SERINE/THREONINE PROTEIN PHOSPHATASE 5 (EC 3.1.3.16)(PP5) (PROTEIN PHOSPHATASE T) (PPT) (FRAGMENT). GENBANK ACCESSION NO:PTN7_RAT PROTEIN-TYROSINE PHOSPHATASE LC-PTP (EC 3.1.3.48)(HEMATOPOIETIC PROTEIN-TYROSINE PHOSPHATASE) (HEPTP). VH01_VACCCVH01_VACCC ID VH01_VACCC STANDARD; PRT; 171 AA. YOPH_YERPS YOPH_YERPSPTN1 ID PTN1_HUMAN STANDARD; PRT; 435 AA. CDC25GI|266561|SP|P30307|MPI3_HUMAN M-PHASE INDUCER PHOSPHATASE 3 (DUALSPECIFICITY PHOSPHATASE CDC25C) CDC14_YEAST GI|6321141|REF|NP_011219.1|SOLUBLE TYROSINE-SPECIFIC PROTEIN PHOSPHATASE; CDC14P [SACCHAROMYCESCEREVISIAE] CDC14B_HUMAN GI|4502699|REF|NP_003662.1| S. CEREVISIAE CDC14HOMOLOG, GENE B [HOMO SAPIENS ] CDC14A_HUMAN GI|4502697|REF|NP_003663.1|S. CEREVISIAE CDC14 HOMOLOG, GENE A [HOMO SAPIENS ]

TABLE V Predicted exons of BMY_HPP4 Exon Start End Sequence 1 7135271414 CTCAGGCAGAACTATGAGGCCAAGAGTGCTCA TGCGCACCAGGCTTTCTTTTTGAAAT TCGAG(SEQ ID NO:11) 2 71577 71667 GAGCTGAAGGAGGTGAGCAAGGAGCAGCCCAGACTGGAGGCTGAGTACCCTGCCAACAC CACCAAGAACTGTTAACCACATGTGCTACCCT (SEQ IDNO:12) 3 71776 71852 ATGACCACTCCAGGGTCAGGCTGACCCAGCTGGAGGGAGAGCCTCATTCTGACTACATCAATGC CAACTTGGTCCCA (SEQ ID NO:13) 4 7288573019 GGCTACACCCGCCCACAGGAGTTCATTGCCTC TCAGGGGCCTCTCAAGAAAACACTGGAGAACTTCTGGCGGCTGGTGCGGGAGCAGCAGGTCCG CATCATCATCATGCCGACCATCAGCATGGAGAACGGGAGG (SEQ ID NO:14) 5 73700 73822 GTGCTGTGTGAGCATTACTGGCTGACCGACTCTACCCCGGACACCCATGGTCACATCACCATCC ACCTCCTAGCTGAGGAGCCTGAGGATGAGTGGACCAAGCGGGAATTCCAGCTGCAGCAC (SEQ ID NO:15) 6 74418 74578GTTGTCCAGCAACATCAACGGAGGGTGGAGCA ACTGCAGTTCACCACCTGATCCGACCACAGCATCCTTGAGGCTCCCAGCTCCCTGCTC GCCTTTATGGAGCTGGTACAGTAGCAGGCAAGGGCCACCCAGGGCGTGGGACCCATCC TGGTGCACTGCAG (SEQ ID NO:16) 7 7470074850 GGGCTGTCCCTGCGGTGTGGGCATGGGCCGGA CAGGCACCTTCGTGGCCCTGTCGAGGCTGCTGCAGCAGCTGGAGGAGGAGCAGATGGTAGACGT GTTCCATGCTGTGTATGCACTCCGGATGCACCAGCCCCTCATGATCCAGACCCTG (SEQ ID NO:17) 8 75210 75277AGCCAGTACGTCTTCCTGCACAGCTGCCTACT GAACAAGATTCTGGAAGGACCCTTCA ACATCTCTGA(SEQ ID NO:18) 9 75407 75494 GTCTTGGCCCATCTCTGTGACGGACCTCCCGCAGGCGTGTGCCAAGAGGGCAGCCAGTGCCAAT GCTGGCTTCTTGAAGGAGTACGAG (SEQ ID NO:19)10 75613 75679 GCCATCAAGGACGAGGCTGGCTTTTCCGCACCCCCGCCTGGCTATGAGCAGGACAGCC CCGTCTCCT (SEQ ID NO:20) 11 75769 75826ATGACCGTTCTCAGGGGCAGTTTTCTCCGGTG GAGGAGAGCCCCCCTGACGACATGCC (SEQ IDNO:21) 12 75960 76119 TCTCTGGAAGCCAATGATCTGTGCTCTGCAGGGTGGGCCCTCTGGCCGTGATCATACG GTGCTGACTGGCCCCGCAGGGCCAAAGGAGCTCTGGGAGCTGGTGTGGCAGCACAGGG CTCATGTGCTTGTCTCTCTTTGCCCACCCAATG TCATGGAGAAG(SEQ ID NO:22) 13 76266 76376 GAATTCTGGCCAACGGAGATGCAGCCCGTAGTCACAGACATGGTGACGGTGCACTGGGTGGCTGAG AGCAGCACAGCAGGCTGGTTCTGTACCCTCCTCAGGGTCACACAT (SEQ ID NO:23) 14 76481 76644GGGGAGAGCAGGAAGGAAAGGGAGGTGCAGAGA CTGCAATTTCCATACCTGGAGCCTGGGCATGAGCTGCCCGCCACCACCCTGCTGCCCTTCCTGGC TGCTGTGGGCCAGTGCTGCTCTCGGGGCAACAACAAGAAGCCGGGCACACTGCTCAGCCACTCCAA (SEQ ID NO:24) 15 76992 77127CAAGGGTGCAACCCAGCTGGGCACCTTCCTGGC CATGGAGCAGCTGCTGCAGCAGGCAGGGTCTGAGTGCACCGTGGATATCTTTAACGTG GCCCTGCAGCAGTCTCAGGCCTGTGGCCTTATGACCCCAACACTG (SEQ ID NO:25) 16 77369 77425AAGCAGTATGTCTACCTCTACAACTGTCTGAAC AGCGCGCTGGCAGACGGGCTGCCC (SEQ IDNO:26)

TABLE VI Internal Left Right RevComp Internal EP Anti- Cloning CloningCloning Cloning EP Sense Sense Gene Primer Primer Primer Primer PrimerPrimer BMY_HPP1 CGCATGGAAGGATTAT CTGTTCGACCAAGCCTGACAATGGATAGCTACTTTTCCTTCCT N/A TACAATTT GCATGACA GGTG(SEQ ID NO:43)CTG (SEQ ID NO:44) GTAAGGCAAATGTCATCACCTTCACCAT CGGATGGA ATGGATAGATCTAGGATAGTAGTAAGAGACGC AGGATTAT CTACTTT (SEQ ID NO:45) (SEQ ID (SEQ IDNO:154) NO:155) ″ TTCGGATGGAAGGATT CTGTTCGACCAAGCCTGACAATGGATAGCTACTTTTCCTTCCT N/A N/A N/A ATGG CTG (SEQ ID NO:47)GTAAGGCAAATGTCATCACCTTCACCAT (SEQ ID NO:46) ATCTAGGATAGTAGTAAGAGACGC(SEQ ID NO:48) BMY_HPP2 CCAACTTCTCCTGGGT CTCCGTCAGGGACACGTGCCGCACGCCCAGGTCCAACAGGAA N/A GAGAAAGC ATGGGAGC GCT (SEQ ID NO:49) CAG(SEQ ID NO:50) CTGGTAGTGGGCGGGGAGCCGCGGCAG AGTCTTCC TAGAGGGTCGCCAGTCCCGCCAGCCGGCCCGGA AGTTCTAC TTAATACT (SEQ ID NO:51) (SEQ ID (SEQID NO:156) NO:157) ″ CAACTTCTCCTGGGTGCT CAGCTGTCGCTGTGACTCCGTCAGGGACACCAGGTGCCGCAC N/A N/A N/A TC(SEQ ID NO:52) GGG (SEQ IDNO:53) GCCCAGGTCCAACAGGAACTGGTAGTG GGCGGGGAOCCGCGOCAGCGCCAGTC (SEQ IDNO:54) BMY_HPP3 CTCCCTGCTTCTGTGGAC AACCTGGATGCTTCCAAAAGAOCAATGTTGTAAGTTGCTTTT N/A N/A N/A AT(SEQ ID NO:55) CTTCT (SEQ IDNO:56) CATACTCTTACTATGGTOOTAACTCCA TCCTGCTTAAGTTCCTGTAAGAATCT (SEQ IDNO:57) ″ TGCTTCTGTGGACATTGC AACCTGGATGCTTCC AAAAGAGCAATGTTGTAAGTTGCTTTTN/A N/A N/A AT(SEQ ID NO:58) CTTCT (SEQ ID NO:59)CATACTCTTACTATGGTGGTAACTCCA TCCTGCTTAAGTTCCTGTAAGAATCT (SEQ ID NO:60)BMY_HPP4 GGCAGAACTATGAGGCCA GACCCTGGAGTGGTC GCTCATGCGCACCAGGCTTTCTTTTTGN/A N/A N/A AG (SEQ ID NO:61) ATAGG (SEQ ID NO:62)AAATTCGAGGAGCTGAAGGAGGTGAGC AAGGAGCAGCCCAGACTGGAGGCTGA (SEQ ID NO:63) ″GCACCAGGCTTTTTTTTG GACCCTGGAGTGGTC TCGAGGAGCTGAAGGAGGTGAGCAAGG N/A N/AN/A A(SEQ ID NO:64) ATAGG (SEQ ID NO:65) AGCAGCCCAGACTGGAGGCTGAGTACCCTGCCAACACCACCAAGAACTGTTAA (SEQ ID NO:66) ″ AGGCAGAACTATGAGGCCGACCCTGGAGTGGTC TCAGCCTCCAGTCTGGGCTGCTCCTTG GCTCATGC N/A N/A AA (SEQ IDNO:71) ATAGG (SEQ ID NO:72) CTCACCTCCTTCAGCTCCTCGAATTTC GCACCAGGAAAAAGAAAGCCTGGTGCGCATGAGC CTTTCTTT (SEQ ID NO:74) TTGAAATT CGAGGAGCTGAAGGAG GTGAGCAA GGAGCAGC CCAGACTG GAGGCTGA (SEQ ID NO:73) BMY_HPP5GGCCAAAGAGCAAACTCA GCATAGCTTGTTGGT N/A N/A ATGGGACC TTATCAGG AG (SEQ IDNO:69) CCCAT (SEQ ID NO:70) AACAAGCT ACTGGTTT ATGC CGG (SEQ ID (SEQ IDNO:67) NO:68)

TABLE VIII Atom Atom Residue No name Residue No X coord Y coord Z coord1 N MET 1 69.582 18.182 8.672 2 CA MET 1 69.395 19.541 8.131 3 C MET 170.570 19.947 7.256 4 O MET 1 70.396 20.201 6.059 5 CB MET 1 69.26920.550 9.267 6 CG MET 1 68.073 20.254 10.160 7 SD MET 1 67.870 21.39211.549 8 CE MET 1 67.694 22.936 10.625 9 N ALA 2 71.766 19.777 7.798 10CA ALA 2 72.997 20.244 7.135 11 C ALA 2 73.470 19.379 5.963 12 O ALA 274.399 19.766 5.248 13 CB ALA 2 74.103 20.321 8.182 14 N ALA 3 72.82718.242 5.755 15 CA ALA 3 73.118 17.415 4.583 16 C ALA 3 72.087 17.6283.469 17 O ALA 3 72.257 17.097 2.366 18 CB ALA 3 73.129 15.952 5.009 19N GLY 4 71.058 18.418 3.754 20 CA GLY 4 69.967 18.671 2.798 21 C GLY 469.309 17.377 2.327 22 O GLY 4 69.228 17.114 1.124 23 N VAL 5 68.79216.606 3.271 24 CA VAL 5 68.290 15.269 2.935 25 C VAL 5 67.085 14.8873.803 26 O VAL 5 66.835 13.713 4.111 27 CB VAL 5 69.467 14.309 3.108 28CG1 VAL 5 69.856 14.148 4.572 29 CG2 VAL 5 69.222 12.952 2.454 30 N LEU6 66.245 15.878 4.045 31 CA LEU 6 65.060 15.698 4.912 32 C LEU 6 64.10514.540 4.542 33 O LEU 6 63.856 13.736 5.454 34 CB LEU 6 64.282 17.0105.011 35 CG LEU 6 64.512 17.720 6.344 36 CD1 LEU 6 64.147 16.806 7.51037 CD2 LEU 6 65.943 18.232 6.500 38 N PRO 7 63.677 14.339 3.292 39 CAPRO 7 62.757 13.220 3.028 40 C PRO 7 63.352 11.816 3.190 41 O PRO 762.579 10.904 3.506 42 CB PRO 7 62.275 13.409 1.625 43 CG PRO 7 63.02714.558 0.983 44 CD PRO 7 63.918 15.128 2.068 45 N GLN 8 64.670 11.6733.259 46 CA GLN 8 65.250 10.338 3.447 47 C GLN 8 65.289 9.903 4.908 48 OGLN 8 65.636 8.751 5.182 49 CB GLN 8 66.654 10.268 2.873 50 CG GLN 866.628 10.270 1.352 51 CD GLN 8 68.031 9.988 0.833 52 OE1 GLN 8 68.45910.546 −0.184 53 NE2 GLN 8 68.740 9.136 1.554 54 N ASN 9 64.901 10.7855.815 55 CA ASN 9 64.698 10.384 7.205 56 C ASN 9 63.244 9.968 7.410 57 OASN 9 62.944 9.083 8.221 58 CB ASN 9 64.990 11.583 8.104 59 CG ASN 966.375 12.157 7.822 60 OD1 ASN 9 67.397 11.480 7.985 61 ND2 ASN 9 66.39213.417 7.424 62 N GLU 10 62.390 10.452 6.522 63 CA GLU 10 60.956 10.1776.614 64 C GLU 10 60.605 8.894 5.876 65 O GLU 10 59.725 8.146 6.320 66CB GLU 10 60.222 11.364 6.002 67 CG GLU 10 60.573 12.649 6.745 68 CD GLU10 60.015 13.867 6.017 69 OE1 GLU 10 60.266 13.966 4.824 70 OE2 GLU 1059.569 14.781 6.701 71 N GLN 11 61.423 8.553 4.896 72 CA GLN 11 61.2727.276 4.185 73 C GLN 11 61.393 6.035 5.090 74 O GLN 11 60.421 5.2725.090 75 CB GLN 11 62.255 7.214 3.018 76 CG GLN 11 61.929 8.252 1.948 77CD GLN 11 60.572 7.967 1.301 78 OE1 GLN 11 60.424 7.000 0.547 79 NE2 GLN11 59.615 8.842 1.563 80 N PRO 12 62.416 5.858 5.928 81 CA PRO 12 62.3964.703 6.838 82 C PRO 12 61.285 4.734 7.899 83 O PRO 12 60.776 3.6558.219 84 CB PRO 12 63.740 4.679 7.498 85 CG PRO 12 64.528 5.909 7.086 86CD PRO 12 63.643 6.651 6.104 87 N TYR 13 60.721 5.892 8.217 88 CA TYR 1359.612 5.912 9.181 89 C TYR 13 58.322 5.470 8.496 90 O TYR 13 57.5894.648 9.058 91 CB TYR 13 59.435 7.317 9.750 92 CG TYR 13 60.630 7.85310.531 93 CD1 TYR 13 60.876 9.220 10.549 94 CD2 TYR 13 61.455 6.98611.238 95 CE1 TYR 13 61.970 9.718 11.243 96 CE2 TYR 13 62.551 7.48211.931 97 CZ TYR 13 62.810 8.846 11.923 98 OH TYR 13 63.964 9.325 12.50599 N SER 14 58.271 5.707 7.194 100 CA SER 14 57.170 5.235 6.347 101 CSER 14 57.337 3.768 5.941 102 O SER 14 56.452 3.205 5.291 103 CB SER 1457.142 6.091 5.085 104 OG SER 14 57.018 7.452 5.473 105 N THR 15 58.4523.163 6.315 106 CA THR 15 58.670 1.739 6.078 107 C THR 15 58.496 0.9517.378 108 O THR 15 58.252 −0.261 7.356 109 CB THR 15 60.094 1.583 5.555110 OG1 THR 15 60.252 2.470 4.457 111 CG2 THR 15 60.388 0.165 5.079 112N LEU 16 58.570 1.658 8.496 113 CA LEU 16 58.401 1.037 9.818 114 C LEU16 56.980 1.194 10.354 115 O LEU 16 56.624 0.571 11.363 116 CB LEU 1659.374 1.694 10.792 117 CG LEU 16 60.826 1.428 10.409 118 CD1 LEU 1661.781 2.252 11.266 119 CD2 LEU 16 61.158 −0.058 10.496 120 N VAL 1756.197 2.008 9.664 121 CA VAL 17 54.788 2.266 9.995 122 C VAL 17 53.9930.972 10.214 123 O VAL 17 54.216 −0.044 9.539 124 CB VAL 17 54.229 3.0718.820 125 CG1 VAL 17 54.351 2.306 7.509 126 CG2 VAL 17 52.795 3.5239.037 127 N ASN 18 53.139 0.982 11.226 128 CA ASN 18 52.379 −0.22311.563 129 C ASN 18 51.049 −0.245 10.820 130 O ASN 18 50.052 0.38611.194 131 CB ASN 18 52.174 −0.298 13.069 132 CG ASN 18 53.534 −0.36213.762 133 OD1 ASN 18 53.935 0.571 14.468 134 ND2 ASN 18 54.237 −1.45913.540 135 N ASN 19 51.075 −1.017 9.750 136 CA ASN 19 49.936 −1.1598.843 137 C ASN 19 48.936 −2.200 9.350 138 O ASN 19 49.078 −3.402 9.100139 CB ASN 19 50.500 −1.593 7.491 140 CG ASN 19 51.685 −0.700 7.116 141OD1 ASN 19 51.561 0.529 7.063 142 ND2 ASN 19 52.818 −1.333 6.852 143 NSER 20 47.949 −1.721 10.088 144 CA SER 20 46.889 −2.590 10.618 145 C SER20 45.830 −2.891 9.562 146 O SER 20 45.525 −2.040 8.721 147 CB SER 2046.232 −1.875 11.801 148 OG SER 20 45.102 −2.630 12.237 149 N GLU 2145.339 −4.120 9.571 150 CA GLU 21 44.151 −4.470 8.780 151 C GLU 2142.990 −3.623 9.296 152 O GLU 21 42.894 −3.409 10.508 153 CB GLU 2143.865 −5.955 8.991 154 CG GLU 21 42.687 −6.464 8.166 155 CD GLU 2142.494 −7.954 8.426 156 OE1 GLU 21 43.469 −8.582 8.815 157 OE2 GLU 2141.380 −8.429 8.263 158 N CYS 22 42.217 −3.047 8.392 159 CA CYS 2241.161 −2.120 8.792 160 C CYS 22 39.765 −2.693 8.569 161 O CYS 22 39.563−3.630 7.786 162 CB CYS 22 41.360 −0.857 7.974 163 SG CYS 22 43.044−0.206 8.046 164 N VAL 23 38.801 −2.093 9.248 165 CA VAL 23 37.410−2.559 9.153 166 C VAL 23 36.644 −1.812 8.059 167 O VAL 23 35.572 −2.2567.621 168 CB VAL 23 36.740 −2.372 10.516 169 CG1 VAL 23 36.590 −0.89910.877 170 CG2 VAL 23 35.391 −3.076 10.594 171 N ALA 24 37.294 −0.8167.480 172 CA ALA 24 36.669 0.010 6.445 173 C ALA 24 36.847 −0.557 5.038174 O ALA 24 36.343 0.014 4.069 175 CB ALA 24 37.252 1.412 6.533 176 NASN 25 37.456 −1.729 4.950 177 CA ASN 25 37.721 −2.366 3.660 178 C ASN25 36.534 −3.178 3.137 179 O ASN 25 36.524 −3.565 1.963 180 CB ASN 2538.930 −3.280 3.838 181 CG ASN 25 40.179 −2.458 4.151 182 OD1 ASN 2540.470 −2.127 5.308 183 ND2 ASN 25 40.901 −2.124 3.099 184 N MET 2635.538 −3.416 3.977 185 CA MET 26 34.328 −4.102 3.504 186 C MET 2633.381 −3.091 2.866 187 O MET 26 33.376 −1.924 3.270 188 CB MET 2633.654 −4.832 4.661 189 CG MET 26 33.177 −3.886 5.754 190 SD MET 2632.426 −4.700 7.181 191 CE MET 26 33.870 −5.620 7.759 192 N LYS 2732.484 −3.577 2.021 193 CA LYS 27 31.626 −2.711 1.187 194 C LYS 2730.792 −1.697 1.974 195 O LYS 27 30.871 −0.498 1.677 196 CB LYS 2730.697 −3.613 0.383 197 CG LYS 27 29.798 −2.809 −0.555 198 CD LYS 2728.844 −3.672 −1.388 199 CE LYS 27 29.482 −4.306 −2.629 200 NZ LYS 2730.336 −5.470 −2.332 201 N GLY 28 30.225 −2.129 3.092 202 CA GLY 2829.458 −1.231 3.969 203 C GLY 28 30.290 −0.040 4.450 204 O GLY 28 29.9961.104 4.083 205 N ASN 29 31.466 −0.337 4.977 206 CA ASN 29 32.335 0.7045.533 207 C ASN 29 33.239 1.388 4.500 208 O ASN 29 33.918 2.358 4.860209 CB ASN 29 33.195 0.102 6.632 210 CG ASN 29 32.343 −0.411 7.789 211OD1 ASN 29 31.243 0.082 8.065 212 ND2 ASN 29 32.880 −1.409 8.463 213 NLEU 30 33.112 1.040 3.226 214 CA LEU 30 33.875 1.715 2.163 215 C LEU 3033.311 3.095 1.834 216 O LEU 30 34.001 3.921 1.228 217 CB LEU 30 33.8390.868 0.893 218 CG LEU 30 34.861 −0.261 0.909 219 CD1 LEU 30 34.663−1.187 −0.285 220 CD2 LEU 30 36.278 0.297 0.916 221 N GLU 31 32.1153.379 2.326 222 CA GLU 31 31.537 4.722 2.198 223 C GLU 31 31.873 5.6163.401 224 O GLU 31 31.357 6.738 3.502 225 CB GLU 31 30.029 4.560 2.047226 CG GLU 31 29.433 3.878 3.269 227 CD GLU 31 28.144 3.156 2.901 228OE1 GLU 31 28.003 2.802 1.740 229 OE2 GLU 31 27.327 2.949 3.791 230 NARG 32 32.699 5.117 4.310 231 CA ARG 32 33.060 5.883 5.506 232 C ARG 3234.325 6.737 5.316 233 O ARG 32 34.177 7.964 5.387 234 CB ARG 32 33.1554.948 6.710 235 CG ARG 32 31.839 4.213 6.909 236 CD ARG 32 31.781 3.4988.249 237 NE ARG 32 30.378 3.247 8.608 238 CZ ARG 32 29.731 3.986 9.510239 NH1 ARG 32 30.398 4.885 10.237 240 NH2 ARG 32 28.440 3.757 9.756 241N PRO 33 35.516 6.182 5.096 242 CA PRO 33 36.646 7.045 4.744 243 C PRO33 36.534 7.511 3.299 244 O PRO 33 36.825 6.765 2.356 245 CB PRO 3337.868 6.204 4.929 246 CG PRO 33 37.438 4.755 5.064 247 CD PRO 33 35.9204.768 5.055 248 N THR 34 36.102 8.749 3.141 249 CA THR 34 35.990 9.3381.799 250 C THR 34 37.323 9.447 1.024 251 O THR 34 37.283 9.160 −0.179252 CB THR 34 35.246 10.673 1.873 253 OG1 THR 34 35.822 11.478 2.892 254CG2 THR 34 33.783 10.455 2.243 255 N PRO 35 38.464 9.823 1.599 256 CAPRO 35 39.706 9.336 0.998 257 C PRO 35 39.844 7.831 1.226 258 O PRO 3540.187 7.406 2.336 259 CB PRO 35 40.802 10.087 1.689 260 CG PRO 3540.226 10.777 2.914 261 CD PRO 35 38.737 10.475 2.893 262 N LYS 3639.863 7.088 0.128 263 CA LYS 36 39.914 5.614 0.169 264 C LYS 36 41.2865.061 0.579 265 O LYS 36 41.397 3.917 1.032 266 CB LYS 36 39.577 5.120−1.237 267 CG LYS 36 39.491 3.599 −1.326 268 CD LYS 36 39.361 3.134−2.772 269 CE LYS 36 38.129 3.728 −3.447 270 NZ LYS 36 38.033 3.283−4.847 271 N TYR 37 42.295 5.915 0.564 272 CA TYR 37 43.631 5.512 1.004273 C TYR 37 43.846 5.752 2.502 274 O TYR 37 44.880 5.347 3.045 275 CBTYR 37 44.681 6.269 0.185 276 CG TYR 37 44.657 7.798 0.284 277 CD1 TYR37 45.380 8.439 1.284 278 CD2 TYR 37 43.942 8.550 −0.642 279 CE1 TYR 3745.363 9.824 1.378 280 CE2 TYR 37 43.923 9.935 −0.550 281 CZ TYR 3744.630 10.568 0.464 282 OH TYR 37 44.587 11.941 0.577 283 N THR 3842.871 6.340 3.176 284 CA THR 38 43.050 6.648 4.596 285 C THR 38 42.2135.707 5.456 286 O THR 38 41.131 6.059 5.941 287 CB THR 38 42.641 8.0944.837 288 OG1 THR 38 43.174 8.881 3.782 289 CG2 THR 38 43.202 8.6096.156 290 N LYS 39 42.750 4.519 5.663 291 CA LYS 39 42.036 3.494 6.428292 C LYS 39 42.502 3.416 7.886 293 O LYS 39 43.693 3.264 8.184 294 CBLYS 39 42.239 2.161 5.716 295 CG LYS 39 41.590 2.151 4.335 296 CD LYS 3940.076 2.292 4.440 297 CE LYS 39 39.405 2.337 3.072 298 NZ LYS 39 39.6621.106 2.313 299 N VAL 40 41.531 3.529 8.777 300 CA VAL 40 41.769 3.42510.225 301 C VAL 40 41.831 1.958 10.671 302 O VAL 40 40.993 1.144 10.260303 CB VAL 40 40.638 4.187 10.919 304 CG1 VAL 40 39.268 3.784 10.385 305CG2 VAL 40 40.691 4.087 12.438 306 N GLY 41 42.816 1.646 11.505 307 CAGLY 41 43.080 0.269 11.961 308 C GLY 41 41.885 −0.455 12.586 309 O GLY41 40.856 0.143 12.912 310 N GLU 42 42.086 −1.735 12.843 311 CA GLU 4240.995 −2.603 13.318 312 C GLU 42 40.700 −2.470 14.817 313 O GLU 4239.903 −1.616 15.224 314 CB GLU 42 41.372 −4.056 13.016 315 CG GLU 4240.208 −5.037 13.176 316 CD GLU 42 39.111 −4.717 12.172 317 OE1 GLU 4239.427 −4.667 10.996 318 OE2 GLU 42 38.001 −4.442 12.611 319 N ARG 4341.515 −3.159 15.603 320 CA ARG 43 41.196 −3.553 16.987 321 C ARG 4341.019 −2.475 18.056 322 O ARG 43 40.698 −1.315 17.771 323 CB ARG 4342.252 −4.560 17.413 324 CG ARG 43 42.024 −5.844 16.627 325 CD ARG 4343.145 −6.861 16.793 326 NE ARG 43 42.810 −8.099 16.069 327 CZ ARG 4343.092 −8.326 14.782 328 NH1 ARG 43 42.660 −9.446 14.197 329 NH2 ARG 4343.743 −7.410 14.061 330 N LEU 44 41.420 −2.857 19.260 331 CA LEU 4440.940 −2.274 20.535 332 C LEU 44 41.307 −0.825 20.889 333 O LEU 4440.872 −0.346 21.941 334 CB LEU 44 41.471 −3.171 21.649 335 CG LEU 4440.988 −4.610 21.499 336 CD1 LEU 44 41.711 −5.534 22.473 337 CD2 LEU 4439.476 −4.712 21.677 338 N ARG 45 42.074 −0.139 20.064 339 CA ARG 4542.359 1.270 20.330 340 C ARG 45 42.277 2.045 19.017 341 O ARG 45 42.4383.271 18.953 342 CB ARG 45 43.741 1.367 20.972 343 CG ARG 45 44.0402.774 21.469 344 CD ARG 45 45.316 2.817 22.295 345 NE ARG 45 45.1432.075 23.552 346 CZ ARG 45 46.140 1.431 24.157 347 NH1 ARG 45 47.3501.403 23.596 348 NH2 ARG 45 45.919 0.787 25.305 349 N HIS 46 41.9041.325 17.978 350 CA HIS 46 41.969 1.898 16.641 351 C HIS 46 40.653 2.58116.302 352 O HIS 46 40.472 3.723 16.742 353 CB HIS 46 42.340 0.80315.655 354 CG HIS 46 43.700 0.165 15.909 355 ND1 HIS 46 44.770 0.74516.483 356 CD2 HIS 46 44.075 −1.114 15.581 357 CE1 HIS 46 45.787 −0.13616.538 358 NE2 HIS 46 45.357 −1.287 15.976 359 N VAL 47 39.754 1.90815.599 360 CA VAL 47 38.471 2.532 15.221 361 C VAL 47 37.658 2.97116.434 362 O VAL 47 37.399 2.178 17.348 363 CB VAL 47 37.650 1.54114.392 364 CG1 VAL 47 36.163 1.879 14.354 365 CG2 VAL 47 38.190 1.43412.977 366 N ILE 48 37.362 4.260 16.470 367 CA ILE 48 36.458 4.82717.472 368 C ILE 48 35.032 4.352 17.216 369 O ILE 48 34.421 4.650 16.181370 CB ILE 48 36.563 6.348 17.385 371 CG1 ILE 48 37.887 6.817 17.971 372CG2 ILE 48 35.404 7.035 18.090 373 CD1 ILE 48 37.969 6.480 19.456 374 NPRO 49 34.540 3.554 18.149 375 CA PRO 49 33.270 2.865 17.959 376 C PRO49 32.085 3.791 18.185 377 O PRO 49 32.083 4.621 19.102 378 CB PRO 4933.270 1.770 18.979 379 CG PRO 49 34.423 1.991 19.946 380 CD PRO 4935.204 3.174 19.399 381 N GLY 50 31.104 3.670 17.312 382 CA GLY 5029.816 4.304 17.576 383 C GLY 50 29.050 3.408 18.537 384 O GLY 50 29.0662.179 18.400 385 N HIS 51 28.351 4.019 19.478 386 CA HIS 51 27.698 3.25420.556 387 C HIS 51 26.280 2.772 20.229 388 O HIS 51 25.346 3.003 21.005389 CB HIS 51 27.673 4.099 21.828 390 CG HIS 51 28.989 4.180 22.588 391ND1 HIS 51 29.133 4.531 23.880 392 CD2 HIS 51 30.253 3.914 22.109 393CE1 HIS 51 30.439 4.492 24.214 394 NE2 HIS 51 31.130 4.112 23.117 395 NMET 52 26.136 2.082 19.109 396 CA MET 52 24.848 1.485 18.743 397 C MET52 25.075 0.169 18.005 398 O MET 52 25.986 0.049 17.177 399 CB MET 5224.018 2.457 17.907 400 CG MET 52 24.634 2.773 16.550 401 SD MET 5223.695 3.953 15.554 402 CE MET 52 22.077 3.150 15.606 403 N ALA 5324.147 −0.754 18.204 404 CA ALA 53 24.284 −2.128 17.691 405 C ALA 5323.978 −2.325 16.202 406 O ALA 53 24.059 −3.457 15.713 407 CB ALA 5323.376 −3.036 18.512 408 N CYS 54 23.645 −1.260 15.491 409 CA CYS 5423.405 −1.382 14.053 410 C CYS 54 24.734 −1.431 13.308 411 O CYS 5425.184 −2.517 12.923 412 CB CYS 54 22.566 −0.201 13.582 413 SG CYS 5420.908 −0.110 14.297 414 N SER 55 25.369 −0.277 13.169 415 CA SER 5526.666 −0.190 12.481 416 C SER 55 27.231 1.228 12.501 417 O SER 5526.638 2.156 11.940 418 CB SER 55 26.511 −0.655 11.034 419 OG SER 5525.477 0.106 10.424 420 N MET 56 28.353 1.384 13.187 421 CA MET 5629.096 2.656 13.196 422 C MET 56 30.600 2.436 13.331 423 O MET 56 31.1732.695 14.400 424 CB MET 56 28.646 3.551 14.348 425 CG MET 56 27.3814.345 14.049 426 SD MET 56 26.900 5.538 15.320 427 CE MET 56 28.3706.588 15.307 428 N ALA 57 31.242 2.052 12.240 429 CA ALA 57 32.706 1.89312.247 430 C ALA 57 33.392 3.145 11.700 431 O ALA 57 33.988 3.123 10.616432 CB ALA 57 33.076 0.686 11.397 433 N CYS 58 33.390 4.185 12.518 434CA CYS 58 33.824 5.524 12.102 435 C CYS 58 35.305 5.618 11.734 436 O CYS58 36.155 4.864 12.227 437 CB CYS 58 33.512 6.469 13.251 438 SG CYS 5831.786 6.444 13.783 439 N GLY 59 35.596 6.575 10.865 440 CA GLY 5936.968 6.800 10.382 441 C GLY 59 37.774 7.717 11.303 442 O GLY 59 38.0078.895 10.997 443 N GLY 60 38.232 7.142 12.401 444 CA GLY 60 39.017 7.88413.390 445 C GLY 60 39.619 6.940 14.424 446 O GLY 60 38.932 6.039 14.914447 N ARG 61 40.895 7.121 14.717 448 CA ARG 61 41.560 6.257 15.705 449 CARG 61 41.929 6.985 16.991 450 O ARG 61 42.244 8.181 16.984 451 CB ARG61 42.771 5.545 15.095 452 CG ARG 61 43.632 6.425 14.197 453 CD ARG 6144.466 7.462 14.939 454 NE ARG 61 45.563 6.879 15.722 455 CZ ARG 6146.833 7.006 15.334 456 NH1 ARG 61 47.823 6.728 16.183 457 NH2 ARG 6147.107 7.616 14.183 458 N ALA 62 41.903 6.251 18.089 459 CA ALA 6242.264 6.837 19.383 460 C ALA 62 43.750 6.672 19.674 461 O ALA 62 44.1935.620 20.148 462 CB ALA 62 41.458 6.156 20.482 463 N CYS 63 44.500 7.74519.497 464 CA CYS 63 45.938 7.694 19.774 465 C CYS 63 46.234 7.93821.252 466 O CYS 63 46.357 9.083 21.706 467 CB CYS 63 46.645 8.74318.931 468 SG CYS 63 48.445 8.739 19.045 469 N LYS 64 46.226 6.84822.001 470 CA LYS 64 46.613 6.860 23.412 471 C LYS 64 48.054 6.38423.552 472 O LYS 64 48.406 5.291 23.091 473 CB LYS 64 45.675 5.92424.169 474 CG LYS 64 46.126 5.674 25.604 475 CD LYS 64 45.182 4.71626.320 476 CE LYS 64 45.706 4.351 27.703 477 NZ LYS 64 47.001 3.66027.607 478 N TYR 65 48.885 7.218 24.151 479 CA TYR 65 50.292 6.84824.356 480 C TYR 65 50.417 5.930 25.573 481 O TYR 65 50.529 6.362 26.725482 CB TYR 65 51.115 8.124 24.463 483 CG TYR 65 51.006 8.959 23.186 484CD1 TYR 65 50.248 10.122 23.169 485 CD2 TYR 65 51.651 8.538 22.030 486CE1 TYR 65 50.138 10.869 22.004 487 CE2 TYR 65 51.545 9.283 20.862 488CZ TYR 65 50.789 10.448 20.853 489 OH TYR 65 50.703 11.202 19.702 490 NGLU 66 50.503 4.650 25.250 491 CA GLU 66 50.281 3.565 26.211 492 C GLU66 51.266 3.423 27.361 493 O GLU 66 52.477 3.638 27.234 494 CB GLU 6650.240 2.244 25.435 495 CG GLU 66 51.547 1.850 24.735 496 CD GLU 6652.510 1.069 25.640 497 OE1 GLU 66 52.038 0.495 26.612 498 OE2 GLU 6653.675 0.978 25.281 499 N ASN 67 50.676 3.106 28.499 500 CA ASN 6751.379 2.400 29.576 501 C ASN 67 50.476 1.416 30.372 502 O ASN 67 50.6311.373 31.597 503 CB ASN 67 51.998 3.439 30.515 504 CG ASN 67 50.9654.467 30.982 505 OD1 ASN 67 49.852 4.121 31.398 506 ND2 ASN 67 51.3425.730 30.899 507 N PRO 68 49.614 0.599 29.756 508 CA PRO 68 48.551−0.034 30.556 509 C PRO 68 48.957 −1.352 31.228 510 O PRO 68 48.432−1.685 32.297 511 CB PRO 68 47.449 −0.296 29.577 512 CG PRO 68 48.006−0.199 28.167 513 CD PRO 68 49.432 0.295 28.323 514 N ALA 69 49.875−2.081 30.617 515 CA ALA 69 50.325 −3.359 31.167 516 C ALA 69 51.279−3.132 32.327 517 O ALA 69 51.904 −2.070 32.438 518 CB ALA 69 51.024−4.157 30.072 519 N ARG 70 51.339 −4.116 33.206 520 CA ARG 70 52.260−4.056 34.338 521 C ARG 70 53.692 −3.993 33.818 522 O ARG 70 54.103−4.803 32.978 523 CB ARG 70 52.044 −5.292 35.204 524 CG ARG 70 52.811−5.206 36.519 525 CD ARG 70 52.437 −6.363 37.438 526 NE ARG 70 50.978−6.406 37.637 527 CZ ARG 70 50.356 −5.922 38.715 528 NH1 ARG 70 51.061−5.383 39.713 529 NH2 ARG 70 49.026 −5.996 38.804 530 N TRP 71 54.353−2.913 34.207 531 CA TRP 71 55.728 −2.595 33.803 532 C TRP 71 55.796−2.191 32.323 533 O TRP 71 56.612 −2.714 31.553 534 CB TRP 71 56.646−3.779 34.113 535 CG TRP 71 58.131 −3.471 34.056 536 CD1 TRP 71 58.826−2.625 34.894 537 CD2 TRP 71 59.090 −3.999 33.115 538 NE1 TRP 71 60.126−2.615 34.511 539 CE2 TRP 71 60.328 −3.422 33.451 540 CE3 TRP 71 58.998−4.885 32.055 541 CZ2 TRP 71 61.460 −3.735 32.710 542 CZ3 TRP 71 60.132−5.193 31.316 543 CH2 TRP 71 61.358 −4.623 31.643 544 N SER 72 54.883−1.327 31.909 545 CA SER 72 55.032 −0.686 30.599 546 C SER 72 55.9850.489 30.750 547 O SER 72 55.906 1.236 31.731 548 CB SER 72 53.687−0.207 30.082 549 OG SER 72 52.882 −1.347 29.831 550 N GLU 73 56.8530.672 29.773 551 CA GLU 73 57.920 1.661 29.944 552 C GLU 73 57.522 3.09329.602 553 O GLU 73 57.643 3.941 30.493 554 CB GLU 73 59.110 1.26829.076 555 CG GLU 73 60.262 2.254 29.257 556 CD GLU 73 61.423 1.89028.339 557 OE1 GLU 73 62.144 0.964 28.684 558 OE2 GLU 73 61.479 2.44327.251 559 N GLN 74 56.848 3.279 28.472 560 CA GLN 74 56.699 4.59227.792 561 C GLN 74 56.706 5.853 28.655 562 O GLN 74 57.733 6.205 29.246563 CB GLN 74 55.438 4.607 26.937 564 CG GLN 74 55.538 3.644 25.759 565CD GLN 74 56.829 3.885 24.980 566 OE1 GLN 74 57.730 3.040 25.007 567 NE2GLN 74 56.924 5.037 24.336 568 N GLU 75 55.709 6.690 28.427 569 CA GLU75 55.673 7.990 29.110 570 C GLU 75 54.257 8.379 29.514 571 O GLU 7553.510 7.587 30.102 572 CB GLU 75 56.253 9.101 28.225 573 CG GLU 7557.772 9.052 28.023 574 CD GLU 75 58.150 8.215 26.800 575 OE1 GLU 7557.252 7.925 26.017 576 OE2 GLU 75 59.327 7.934 26.630 577 N GLN 7653.950 9.642 29.269 578 CA GLN 76 52.644 10.211 29.608 579 C GLN 7651.567 9.679 28.670 580 O GLN 76 51.692 9.778 27.444 581 CB GLN 7652.706 11.737 29.491 582 CG GLN 76 53.713 12.381 30.447 583 CD GLN 7655.051 12.678 29.763 584 OE1 GLN 76 55.341 12.161 28.674 585 NE2 GLN 7655.886 13.421 30.467 586 N ALA 77 50.454 9.272 29.259 587 CA ALA 7749.352 8.659 28.498 588 C ALA 77 48.325 9.656 27.966 589 O ALA 77 47.1299.543 28.258 590 CB ALA 77 48.647 7.641 29.385 591 N ILE 78 48.78310.610 27.174 592 CA ILE 78 47.863 11.583 26.584 593 C ILE 78 47.15910.969 25.373 594 O ILE 78 47.630 9.975 24.802 595 CB ILE 78 48.61712.867 26.257 596 CG1 ILE 78 49.834 12.617 25.382 597 CG2 ILE 78 49.03413.570 27.545 598 CD1 ILE 78 50.571 13.918 25.084 599 N LYS 79 45.94411.438 25.134 600 CA LYS 79 45.081 10.825 24.115 601 C LYS 79 44.48111.830 23.126 602 O LYS 79 43.805 12.794 23.512 603 CB LYS 79 43.93710.128 24.838 604 CG LYS 79 44.406 9.091 25.850 605 CD LYS 79 43.2128.464 26.555 606 CE LYS 79 42.351 9.537 27.213 607 NZ LYS 79 41.1508.950 27.826 608 N GLY 80 44.633 11.515 21.851 609 CA GLY 80 44.03612.335 20.784 610 C GLY 80 43.501 11.483 19.632 611 O GLY 80 44.21210.640 19.078 612 N VAL 81 42.248 11.699 19.275 613 CA VAL 81 41.64510.946 18.169 614 C VAL 81 41.923 11.618 16.828 615 O VAL 81 41.52912.766 16.605 616 CB VAL 81 40.139 10.838 18.392 617 CG1 VAL 81 39.42810.202 17.201 618 CG2 VAL 81 39.835 10.047 19.656 619 N TYR 82 42.61610.905 15.955 620 CA TYR 82 42.869 11.403 14.593 621 C TYR 82 41.64211.074 13.759 622 O TYR 82 41.294 9.897 13.614 623 CB TYR 82 44.07710.717 13.952 624 CG TYR 82 45.487 10.949 14.516 625 CD1 TYR 82 45.73211.013 15.882 626 CD2 TYR 82 46.546 11.062 13.624 627 CE1 TYR 82 47.02011.204 16.355 628 CE2 TYR 82 47.837 11.255 14.094 629 CZ TYR 82 48.07011.324 15.461 630 OH TYR 82 49.348 11.512 15.934 631 N SER 83 40.98012.095 13.251 632 CA SER 83 39.709 11.877 12.560 633 C SER 83 39.68112.397 11.126 634 O SER 83 40.202 13.473 10.793 635 CB SER 83 38.62612.573 13.362 636 OG SER 83 38.754 12.154 14.715 637 N SER 84 38.97911.633 10.307 638 CA SER 84 38.657 12.044 8.941 639 C SER 84 37.61413.156 8.975 640 O SER 84 37.138 13.562 10.045 641 CB SER 84 38.11610.851 8.165 642 OG SER 84 39.118 9.845 8.164 643 N TRP 85 37.338 13.7187.815 644 CA TRP 85 36.427 14.855 7.758 645 C TRP 85 34.976 14.424 7.836646 O TRP 85 34.582 13.351 7.365 647 CB TRP 85 36.711 15.752 6.550 648CG TRP 85 36.868 15.131 5.172 649 CD1 TRP 85 37.923 14.370 4.729 650 CD2TRP 85 35.964 15.264 4.047 651 NE1 TRP 85 37.710 14.038 3.432 652 CE2TRP 85 36.556 14.558 2.987 653 CE3 TRP 85 34.763 15.926 3.868 654 CZ2TRP 85 35.916 14.509 1.754 655 CZ3 TRP 85 34.132 15.877 2.628 656 CH2TRP 85 34.706 15.169 1.578 657 N VAL 86 34.220 15.241 8.548 658 CA VAL86 32.795 15.003 8.784 659 C VAL 86 31.962 15.331 7.540 660 O VAL 8631.477 16.446 7.312 661 CB VAL 86 32.410 15.833 10.005 662 CG1 VAL 8632.857 17.276 9.871 663 CG2 VAL 86 30.934 15.752 10.347 664 N THR 8731.913 14.332 6.679 665 CA THR 87 31.179 14.425 5.422 666 C THR 8729.687 14.290 5.708 667 O THR 87 29.264 13.423 6.478 668 CB THR 8731.674 13.310 4.495 669 OG1 THR 87 33.097 13.322 4.485 670 CG2 THR 8731.195 13.474 3.056 671 N ASP 88 28.901 15.089 5.003 672 CA ASP 8827.435 15.148 5.153 673 C ASP 88 26.700 13.962 4.503 674 O ASP 88 25.47313.854 4.604 675 CB ASP 88 27.005 16.467 4.503 676 CG ASP 88 25.50116.716 4.588 677 OD1 ASP 88 24.837 16.511 3.580 678 OD2 ASP 88 25.06517.212 5.614 679 N ASN 89 27.451 13.050 3.908 680 CA ASN 89 26.88611.881 3.225 681 C ASN 89 26.010 11.001 4.126 682 O ASN 89 26.427 10.5145.179 683 CB ASN 89 28.025 11.060 2.598 684 CG ASN 89 29.028 10.4313.582 685 OD1 ASN 89 29.207 10.861 4.730 686 ND2 ASN 89 29.677 9.3883.093 687 N ILE 90 24.732 10.991 3.785 688 CA ILE 90 23.753 10.066 4.369689 C ILE 90 23.098 9.270 3.239 690 O ILE 90 22.384 8.280 3.459 691 CBILE 90 22.734 10.894 5.156 692 CG1 ILE 90 21.559 10.056 5.653 693 CG2ILE 90 22.253 12.084 4.333 694 CD1 ILE 90 20.503 10.892 6.367 695 N LEU91 23.611 9.539 2.050 696 CA LEU 91 23.026 9.037 0.801 697 C LEU 9123.152 7.529 0.577 698 O LEU 91 22.277 6.964 −0.087 699 CB LEU 91 23.7519.752 −0.331 700 CG LEU 91 23.219 9.334 −1.695 701 CD1 LEU 91 21.7589.741 −1.853 702 CD2 LEU 91 24.069 9.925 −2.811 703 N ALA 92 24.0136.855 1.320 704 CA ALA 92 24.203 5.419 1.115 705 C ALA 92 23.143 4.5731.822 706 O ALA 92 23.045 3.369 1.563 707 CB ALA 92 25.588 5.058 1.618708 N MET 93 22.324 5.212 2.643 709 CA MET 93 21.169 4.543 3.241 710 CMET 93 19.932 4.726 2.359 711 O MET 93 18.919 4.037 2.525 712 CB MET 9320.936 5.176 4.605 713 CG MET 93 19.904 4.421 5.430 714 SD MET 93 19.5485.143 7.045 715 CE MET 93 19.043 6.790 6.496 716 N ALA 94 20.034 5.6351.403 717 CA ALA 94 18.959 5.817 0.426 718 C ALA 94 19.291 4.973 −0.795719 O ALA 94 18.406 4.452 −1.485 720 CB ALA 94 18.884 7.287 0.035 721 NARG 95 20.584 4.847 −1.037 722 CA ARG 95 21.078 3.858 −1.985 723 C ARG95 20.910 2.497 −1.330 724 O ARG 95 21.093 2.377 −0.115 725 CB ARG 9522.534 4.160 −2.316 726 CG ARG 95 22.641 5.568 −2.884 727 CD ARG 9523.788 5.689 −3.879 728 NE ARG 95 23.556 4.782 −5.017 729 CZ ARG 9522.886 5.121 −6.122 730 NH1 ARG 95 22.469 6.378 −6.300 731 NH2 ARG 9522.697 4.217 −7.086 732 N PRO 96 20.634 1.485 −2.135 733 CA PRO 9619.763 0.377 −1.695 734 C PRO 96 20.355 −0.654 −0.724 735 O PRO 9619.710 −1.685 −0.514 736 CB PRO 96 19.348 −0.321 −2.954 737 CG PRO 9620.006 0.337 −4.153 738 CD PRO 96 20.759 1.531 −3.598 739 N SER 9721.524 −0.428 −0.146 740 CA SER 97 22.109 −1.500 0.656 741 C SER 9723.013 −1.054 1.802 742 O SER 97 23.704 −1.922 2.351 743 CB SER 9722.930 −2.386 −0.268 744 OG SER 97 24.007 −1.596 −0.754 745 N SER 9823.082 0.223 2.146 746 CA SER 98 24.019 0.556 3.226 747 C SER 98 23.6091.676 4.187 748 O SER 98 22.446 1.818 4.582 749 CB SER 98 25.400 0.7932.622 750 OG SER 98 25.275 1.682 1.527 751 N GLU 99 24.631 2.341 4.700752 CA GLU 99 24.506 3.173 5.903 753 C GLU 99 24.464 4.683 5.668 754 OGLU 99 24.930 5.222 4.655 755 CB GLU 99 25.728 2.848 6.757 756 CG GLU 9925.804 1.351 7.039 757 CD GLU 99 27.232 0.924 7.370 758 OE1 GLU 9927.830 0.307 6.500 759 OE2 GLU 99 27.578 0.974 8.541 760 N LEU 10023.825 5.346 6.616 761 CA LEU 100 23.952 6.802 6.752 762 C LEU 10025.328 7.047 7.355 763 O LEU 100 25.861 6.148 8.016 764 CB LEU 10022.856 7.388 7.649 765 CG LEU 100 23.035 7.149 9.152 766 CD1 LEU 10022.368 8.257 9.958 767 CD2 LEU 100 22.547 5.778 9.621 768 N LEU 10125.945 8.179 7.071 769 CA LEU 101 27.336 8.312 7.496 770 C LEU 10127.658 9.477 8.434 771 O LEU 101 26.914 9.821 9.364 772 CB LEU 10128.233 8.340 6.267 773 CG LEU 101 29.157 7.127 6.178 774 CD1 LEU 10130.092 7.081 7.376 775 CD2 LEU 101 28.386 5.820 6.051 776 N GLU 10228.772 10.116 8.124 777 CA GLU 102 29.608 10.691 9.182 778 C GLU 10229.334 12.107 9.659 779 O GLU 102 29.966 12.491 10.651 780 CB GLU 10231.077 10.507 8.808 781 CG GLU 102 31.374 10.810 7.345 782 CD GLU 10232.824 10.443 7.033 783 OE1 GLU 102 33.377 9.651 7.786 784 OE2 GLU 10233.352 10.968 6.062 785 N LYS 103 28.309 12.788 9.177 786 CA LYS 10328.083 14.123 9.735 787 C LYS 103 27.446 14.019 11.115 788 O LYS 10327.920 14.654 12.064 789 CB LYS 103 27.207 14.984 8.839 790 CG LYS 10327.417 16.443 9.233 791 CD LYS 103 26.409 17.389 8.599 792 CE LYS 10325.011 17.138 9.149 793 NZ LYS 103 24.057 18.130 8.630 794 N TYR 10426.600 13.016 11.278 795 CA TYR 104 25.983 12.783 12.581 796 C TYR 10426.847 11.854 13.428 797 O TYR 104 26.937 12.032 14.651 798 CB TYR 10424.619 12.148 12.340 799 CG TYR 104 23.863 11.778 13.610 800 CD1 TYR 10423.700 12.715 14.624 801 CD2 TYR 104 23.323 10.505 13.743 802 CE1 TYR104 23.019 12.370 15.783 803 CE2 TYR 104 22.642 10.158 14.902 804 CZ TYR104 22.499 11.090 15.922 805 OH TYR 104 21.954 10.703 17.125 806 N HIS105 27.701 11.097 12.760 807 CA HIS 105 28.493 10.095 13.471 808 C HIS105 29.681 10.699 14.206 809 O HIS 105 29.883 10.316 15.364 810 CB HIS105 28.944 9.013 12.498 811 CG HIS 105 27.816 8.119 12.014 812 ND1 HIS105 27.909 7.152 11.083 813 CD2 HIS 105 26.512 8.119 12.455 814 CE1 HIS105 26.704 6.573 10.918 815 NE2 HIS 105 25.839 7.171 11.766 816 N ILE106 30.159 11.847 13.745 817 CA ILE 106 31.229 12.545 14.472 818 C ILE106 30.692 13.214 15.741 819 O ILE 106 31.338 13.133 16.796 820 CB ILE106 31.845 13.588 13.542 821 CG1 ILE 106 32.562 12.932 12.366 822 CG2ILE 106 32.812 14.492 14.298 823 CD1 ILE 106 33.820 12.190 12.803 824 NILE 107 29.409 13.544 15.720 825 CA ILE 107 28.757 14.124 16.896 826 CILE 107 28.391 13.035 17.903 827 O ILE 107 28.568 13.223 19.115 828 CBILE 107 27.496 14.835 16.422 829 CG1 ILE 107 27.836 15.847 15.334 830CG2 ILE 107 26.792 15.522 17.587 831 CD1 ILE 107 26.584 16.538 14.806832 N ASP 108 28.180 11.833 17.390 833 CA ASP 108 27.923 10.676 18.249834 C ASP 108 29.208 10.193 18.914 835 O ASP 108 29.172 9.807 20.087 836CB ASP 108 27.365 9.538 17.402 837 CG ASP 108 26.050 9.928 16.738 838OD1 ASP 108 25.815 9.451 15.634 839 OD2 ASP 108 25.258 10.599 17.385 840N GLN 109 30.342 10.437 18.277 841 CA GLN 109 31.632 10.092 18.881 842 CGLN 109 31.991 11.082 19.984 843 O GLN 109 32.399 10.653 21.075 844 CBGLN 109 32.690 10.135 17.788 845 CG GLN 109 32.379 9.125 16.692 846 CDGLN 109 33.233 9.416 15.463 847 OE1 GLN 109 32.718 9.563 14.346 848 NE2GLN 109 34.531 9.522 15.686 849 N PHE 110 31.571 12.327 19.803 850 CAPHE 110 31.764 13.353 20.834 851 C PHE 110 31.020 12.981 22.104 852 OPHE 110 31.659 12.752 23.142 853 CB PHE 110 31.206 14.694 20.367 854 CGPHE 110 31.950 15.394 19.238 855 CD1 PHE 110 31.252 16.247 18.393 856CD2 PHE 110 33.314 15.208 19.065 857 CE1 PHE 110 31.914 16.901 17.366858 CE2 PHE 110 33.979 15.862 18.037 859 CZ PHE 110 33.277 16.708 17.188860 N LEU 111 29.754 12.635 21.938 861 CA LEU 111 28.891 12.344 23.087862 C LEU 111 29.102 10.953 23.688 863 O LEU 111 28.781 10.751 24.864864 CB LEU 111 27.447 12.460 22.614 865 CG LEU 111 27.138 13.856 22.083866 CD1 LEU 111 25.781 13.888 21.388 867 CD2 LEU 111 27.209 14.90223.191 868 N SER 112 29.753 10.062 22.961 869 CA SER 112 29.974 8.70923.478 870 C SER 112 31.297 8.564 24.222 871 O SER 112 31.474 7.59524.970 872 CB SER 112 29.958 7.730 22.309 873 OG SER 112 31.072 8.00621.468 874 N HIS 113 32.216 9.496 24.029 875 CA HIS 113 33.502 9.38724.724 876 C HIS 113 33.821 10.601 25.588 877 O HIS 113 34.852 10.62426.274 878 CB HIS 113 34.588 9.175 23.680 879 CG HIS 113 34.422 7.87522.918 880 ND1 HIS 113 34.259 6.654 23.459 881 CD2 HIS 113 34.398 7.71821.554 882 CE1 HIS 113 34.146 5.742 22.472 883 NE2 HIS 113 34.227 6.40121.296 884 N GLY 114 32.968 11.609 25.517 885 CA GLY 114 33.180 12.83626.288 886 C GLY 114 34.321 13.619 25.659 887 O GLY 114 35.222 14.10526.355 888 N ILE 115 34.289 13.676 24.339 889 CA ILE 115 35.368 14.28023.557 890 C ILE 115 35.398 15.792 23.743 891 O ILE 115 34.365 16.46523.626 892 CB ILE 115 35.115 13.933 22.092 893 CG1 ILE 115 35.139 12.42321.876 894 CG2 ILE 115 36.122 14.601 21.164 895 CD1 ILE 115 36.53811.849 22.044 896 N LYS 116 36.582 16.316 24.018 897 CA LYS 116 36.75317.768 24.159 898 C LYS 116 36.915 18.452 22.807 899 O LYS 116 38.01418.875 22.439 900 CB LYS 116 37.950 18.072 25.052 901 CG LYS 116 37.52818.042 26.515 902 CD LYS 116 36.401 19.047 26.742 903 CE LYS 116 35.95419.087 28.197 904 NZ LYS 116 34.842 20.032 28.385 905 N THR 117 35.77118.654 22.162 906 CA THR 117 35.621 19.299 20.844 907 C THR 117 36.64818.926 19.776 908 O THR 117 37.519 18.053 19.938 909 CB THR 117 35.54120.816 21.002 910 OG1 THR 117 36.584 21.254 21.862 911 CG2 THR 11734.221 21.237 21.635 912 N ILE 118 36.417 19.519 18.621 913 CA ILE 11837.154 19.154 17.415 914 C ILE 118 38.293 20.137 17.125 915 O ILE 11838.134 21.365 17.127 916 CB ILE 118 36.111 19.041 16.300 917 CG1 ILE 11836.394 17.862 15.387 918 CG2 ILE 118 35.967 20.316 15.472 919 CD1 ILE118 35.199 17.607 14.475 920 N ILE 119 39.479 19.570 17.023 921 CA ILE119 40.674 20.350 16.721 922 C ILE 119 40.793 20.532 15.219 923 O ILE119 40.752 19.571 14.442 924 CB ILE 119 41.914 19.650 17.272 925 CG1 ILE119 41.943 19.687 18.787 926 CG2 ILE 119 43.203 20.255 16.732 927 CD1ILE 119 43.309 19.252 19.300 928 N ASN 120 40.887 21.793 14.849 929 CAASN 120 41.073 22.224 13.474 930 C ASN 120 42.289 21.600 12.809 931 OASN 120 43.225 21.128 13.468 932 CB ASN 120 41.324 23.726 13.520 933 CGASN 120 42.587 24.064 14.330 934 OD1 ASN 120 43.722 23.857 13.883 935ND2 ASN 120 42.373 24.726 15.450 936 N LEU 121 42.237 21.568 11.492 937CA LEU 121 43.450 21.309 10.725 938 C LEU 121 43.658 22.383 9.669 939 OLEU 121 44.718 23.017 9.638 940 CB LEU 121 43.401 19.937 10.078 941 CGLEU 121 43.758 18.834 11.061 942 CD1 LEU 121 43.749 17.491 10.352 943CD2 LEU 121 45.124 19.094 11.686 944 N GLN 122 42.658 22.588 8.826 945CA GLN 122 42.776 23.597 7.762 946 C GLN 122 41.405 24.198 7.420 947 OGLN 122 40.431 23.956 8.148 948 CB GLN 122 43.472 22.918 6.571 949 CGGLN 122 42.637 22.364 5.408 950 CD GLN 122 41.586 21.316 5.773 951 OE1GLN 122 40.551 21.635 6.372 952 NE2 GLN 122 41.814 20.107 5.301 953 NARG 123 41.383 25.111 6.459 954 CA ARG 123 40.112 25.569 5.888 955 C ARG123 39.445 24.433 5.122 956 O ARG 123 40.049 23.849 4.213 957 CB ARG 12340.393 26.662 4.872 958 CG ARG 123 41.253 27.780 5.428 959 CD ARG 12341.601 28.783 4.336 960 NE ARG 123 42.307 28.108 3.234 961 CZ ARG 12341.813 28.011 1.997 962 NH1 ARG 123 40.617 28.532 1.710 963 NH2 ARG 12342.510 27.382 1.049 964 N PRO 124 38.145 24.301 5.323 965 CA PRO 12437.364 23.270 4.624 966 C PRO 124 37.193 23.506 3.115 967 O PRO 12437.145 22.529 2.352 968 CB PRO 124 36.025 23.312 5.298 969 CG PRO 12435.971 24.462 6.290 970 CD PRO 124 37.338 25.117 6.238 971 N GLY 12537.365 24.749 2.681 972 CA GLY 125 37.105 25.138 1.286 973 C GLY 12538.355 25.187 0.405 974 O GLY 125 38.711 26.232 −0.149 975 N GLU 12638.986 24.037 0.269 976 CA GLU 126 40.101 23.843 −0.667 977 C GLU 12639.916 22.450 −1.249 978 O GLU 126 40.518 21.484 −0.769 979 CB GLU 12641.447 23.985 0.044 980 CG GLU 126 41.418 23.386 1.443 981 CD GLU 12642.827 23.187 1.989 982 OE1 GLU 126 43.143 22.054 2.330 983 OE2 GLU 12643.529 24.172 2.170 984 N HIS 127 39.086 22.388 −2.284 985 CA HIS 12738.432 21.139 −2.715 986 C HIS 127 37.507 20.715 −1.578 987 O HIS 12737.160 21.537 −0.720 988 CB HIS 127 39.428 20.020 −3.031 989 CG HIS 12740.524 20.386 −4.013 990 ND1 HIS 127 40.380 20.673 −5.321 991 CD2 HIS127 41.867 20.482 −3.730 992 CE1 HIS 127 41.588 20.951 −5.853 993 NE2HIS 127 42.507 20.833 −4.867 994 N ALA 128 37.057 19.473 −1.596 995 CAALA 128 36.279 18.949 −0.457 996 C ALA 128 37.239 18.486 0.640 997 O ALA128 37.448 17.284 0.848 998 CB ALA 128 35.421 17.786 −0.938 999 N SER129 37.783 19.449 1.364 1000 CA SER 129 38.948 19.170 2.196 1001 C SER129 38.606 18.981 3.665 1002 O SER 129 39.463 18.541 4.443 1003 CB SER129 39.899 20.341 2.036 1004 OG SER 129 41.207 19.906 2.364 1005 N CYS130 37.383 19.319 4.035 1006 CA CYS 130 36.922 19.121 5.411 1007 C CYS130 35.443 19.471 5.489 1008 O CYS 130 35.022 20.499 4.954 1009 CB CYS130 37.717 20.028 6.350 1010 SG CYS 130 37.778 19.479 8.067 1011 N GLY131 34.661 18.613 6.116 1012 CA GLY 131 33.228 18.891 6.272 1013 C GLY131 32.962 19.833 7.445 1014 O GLY 131 33.828 20.030 8.306 1015 N ASN132 31.771 20.409 7.462 1016 CA ASN 132 31.397 21.326 8.548 1017 C ASN132 30.022 21.008 9.141 1018 O ASN 132 28.984 21.206 8.499 1019 CB ASN132 31.402 22.761 8.028 1020 CG ASN 132 30.993 23.686 9.170 1021 OD1 ASN132 31.421 23.490 10.313 1022 ND2 ASN 132 30.109 24.621 8.876 1023 N PRO133 30.034 20.467 10.351 1024 CA PRO 133 28.805 20.288 11.135 1025 C PRO133 28.365 21.525 11.944 1026 O PRO 133 27.252 21.530 12.484 1027 CB PRO133 29.169 19.190 12.086 1028 CG PRO 133 30.689 19.125 12.189 1029 CDPRO 133 31.220 20.045 11.100 1030 N LEU 134 29.197 22.552 12.020 1031 CALEU 134 28.928 23.682 12.914 1032 C LEU 134 28.041 24.744 12.279 1033 OLEU 134 28.259 25.185 11.141 1034 CB LEU 134 30.260 24.310 13.315 1035CG LEU 134 31.145 23.316 14.065 1036 CD1 LEU 134 32.514 23.914 14.3701037 CD2 LEU 134 30.473 22.846 15.350 1038 N GLU 135 27.052 25.16613.048 1039 CA GLU 135 26.172 26.258 12.626 1040 C GLU 135 26.925 27.58112.758 1041 O GLU 135 27.237 28.036 13.864 1042 CB GLU 135 24.932 26.22513.514 1043 CG GLU 135 23.821 27.140 13.016 1044 CD GLU 135 22.54926.877 13.820 1045 OE1 GLU 135 22.673 26.118 14.775 1046 OE2 GLU 13521.489 27.082 13.244 1047 N GLN 136 27.229 28.168 11.611 1048 CA GLN 13628.077 29.366 11.560 1049 C GLN 136 27.301 30.640 11.864 1050 O GLN 13627.862 31.618 12.369 1051 CB GLN 136 28.643 29.459 10.150 1052 CG GLN136 29.423 28.203 9.787 1053 CD GLN 136 29.725 28.195 8.293 1054 OE1 GLN136 29.167 27.387 7.541 1055 NE2 GLN 136 30.573 29.120 7.877 1056 N GLU137 26.014 30.614 11.574 1057 CA GLU 137 25.150 31.722 11.974 1058 C GLU137 24.177 31.221 13.030 1059 O GLU 137 23.581 30.149 12.864 1060 CB GLU137 24.418 32.291 10.762 1061 CG GLU 137 23.632 31.233 10.000 1062 CDGLU 137 22.867 31.887 8.855 1063 OE1 GLU 137 22.781 31.265 7.805 1064OE2 GLU 137 22.472 33.032 9.020 1065 N SER 138 24.060 31.986 14.104 1066CA SER 138 23.226 31.604 15.250 1067 C SER 138 23.669 30.259 15.822 1068O SER 138 23.109 29.207 15.490 1069 CB SER 138 21.759 31.541 14.830 1070OG SER 138 21.003 31.101 15.948 1071 N GLY 139 24.724 30.304 16.614 1072CA GLY 139 25.233 29.096 17.258 1073 C GLY 139 25.932 29.478 18.553 1074O GLY 139 25.311 30.001 19.485 1075 N PHE 140 27.227 29.235 18.587 1076CA PHE 140 28.023 29.597 19.759 1077 C PHE 140 28.840 30.846 19.478 1078O PHE 140 28.592 31.552 18.495 1079 CB PHE 140 28.918 28.428 20.147 1080CG PHE 140 28.135 27.244 20.707 1081 CD1 PHE 140 27.682 27.283 22.0191082 CD2 PHE 140 27.860 26.139 19.910 1083 CE1 PHE 140 26.966 26.21222.539 1084 CE2 PHE 140 27.144 25.069 20.430 1085 CZ PHE 140 26.69925.104 21.745 1086 N THR 141 29.746 31.146 20.391 1087 CA THR 141 30.61532.321 20.264 1088 C THR 141 31.653 32.097 19.162 1089 O THR 141 31.85330.956 18.731 1090 CB THR 141 31.286 32.549 21.615 1091 OG1 THR 14131.941 33.806 21.601 1092 CG2 THR 141 32.300 31.460 21.949 1093 N TYR142 32.113 33.186 18.565 1094 CA TYR 142 33.120 33.121 17.492 1095 C TYR142 34.209 34.181 17.682 1096 O TYR 142 33.982 35.362 17.393 1097 CB TYR142 32.444 33.394 16.145 1098 CG TYR 142 31.164 32.611 15.854 1099 CD1TYR 142 29.955 33.292 15.775 1100 CD2 TYR 142 31.204 31.238 15.644 1101CE1 TYR 142 28.781 32.597 15.517 1102 CE2 TYR 142 30.029 30.541 15.3881103 CZ TYR 142 28.821 31.223 15.331 1104 OH TYR 142 27.647 30.53115.136 1105 N LEU 143 35.382 33.759 18.123 1106 CA LEU 143 36.538 34.66718.266 1107 C LEU 143 37.080 35.096 16.903 1108 O LEU 143 37.544 34.25516.125 1109 CB LEU 143 37.626 33.929 19.044 1110 CG LEU 143 38.90934.741 19.216 1111 CD1 LEU 143 38.692 35.937 20.135 1112 CD2 LEU 14340.028 33.864 19.764 1113 N PRO 144 37.040 36.395 16.638 1114 CA PRO 14437.290 36.941 15.294 1115 C PRO 144 38.764 37.165 14.918 1116 O PRO 14439.135 38.289 14.563 1117 CB PRO 144 36.570 38.254 15.289 1118 CG PRO144 36.205 38.629 16.718 1119 CD PRO 144 36.580 37.430 17.569 1120 N GLU145 39.605 36.149 15.032 1121 CA GLU 145 40.963 36.277 14.489 1122 C GLU145 40.975 35.718 13.074 1123 O GLU 145 40.394 34.654 12.829 1124 CB GLU145 41.981 35.541 15.345 1125 CG GLU 145 42.179 36.208 16.698 1126 CDGLU 145 43.285 35.485 17.462 1127 OE1 GLU 145 44.016 34.732 16.832 1128OE2 GLU 145 43.291 35.596 18.681 1129 N ALA 146 41.745 36.358 12.2051130 CA ALA 146 41.772 36.020 10.769 1131 C ALA 146 41.935 34.533 10.4831132 O ALA 146 40.993 33.877 10.028 1133 CB ALA 146 42.932 36.756 10.1231134 N PHE 147 43.069 33.979 10.873 1135 CA PHE 147 43.301 32.550 10.6401136 C PHE 147 42.961 31.685 11.856 1137 O PHE 147 43.265 30.485 11.8461138 CB PHE 147 44.743 32.302 10.203 1139 CG PHE 147 45.104 32.760 8.7831140 CD1 PHE 147 44.119 33.164 7.890 1141 CD2 PHE 147 46.434 32.7538.382 1142 CE1 PHE 147 44.463 33.575 6.609 1143 CE2 PHE 147 46.78033.163 7.101 1144 CZ PHE 147 45.795 33.577 6.215 1145 N MET 148 42.31632.267 12.857 1146 CA MET 148 42.010 31.551 14.106 1147 C MET 148 40.65231.969 14.670 1148 O MET 148 40.578 32.723 15.652 1149 CB MET 148 43.08131.840 15.157 1150 CG MET 148 44.417 31.178 14.841 1151 SD MET 14845.728 31.446 16.054 1152 CE MET 148 47.004 30.406 15.307 1153 N GLU 14939.594 31.418 14.102 1154 CA GLU 149 38.246 31.738 14.593 1155 C GLU 14937.769 30.699 15.601 1156 O GLU 149 37.243 29.646 15.223 1157 CB GLU 14937.257 31.790 13.435 1158 CG GLU 149 35.869 32.194 13.924 1159 CD GLU149 34.845 32.048 12.803 1160 OE1 GLU 149 33.686 31.814 13.118 1161 OE2GLU 149 35.250 32.129 11.652 1162 N ALA 150 37.921 31.009 16.875 1163 CAALA 150 37.520 30.058 17.921 1164 C ALA 150 36.026 30.099 18.231 1165 OALA 150 35.539 31.038 18.872 1166 CB ALA 150 38.307 30.354 19.192 1167 NGLY 151 35.325 29.061 17.810 1168 CA GLY 151 33.903 28.917 18.129 1169 CGLY 151 33.774 28.180 19.456 1170 O GLY 151 34.010 28.752 20.529 1171 NILE 152 33.405 26.911 19.373 1172 CA ILE 152 33.506 26.032 20.544 1173 CILE 152 34.898 25.412 20.573 1174 O ILE 152 35.379 24.937 21.608 1175 CBILE 152 32.429 24.951 20.513 1176 CG1 ILE 152 32.175 24.424 19.105 1177CG2 ILE 152 31.146 25.457 21.154 1178 CD1 ILE 152 31.091 23.354 19.1031179 N TYR 153 35.500 25.380 19.396 1180 CA TYR 153 36.934 25.163 19.2531181 C TYR 153 37.399 25.983 18.059 1182 O TYR 153 36.591 26.481 17.2661183 CB TYR 153 37.281 23.689 19.105 1184 CG TYR 153 38.254 23.21120.184 1185 CD1 TYR 153 38.314 23.882 21.401 1186 CD2 TYR 153 39.08022.118 19.957 1187 CE1 TYR 153 39.184 23.452 22.393 1188 CE2 TYR 15339.952 21.687 20.948 1189 CZ TYR 153 40.001 22.353 22.164 1190 OH TYR153 40.847 21.907 23.155 1191 N PHE 154 38.697 26.194 18.010 1192 CA PHE154 39.306 27.134 17.065 1193 C PHE 154 39.332 26.579 15.648 1194 O PHE154 39.605 25.390 15.453 1195 CB PHE 154 40.737 27.401 17.532 1196 CGPHE 154 41.024 26.975 18.976 1197 CD1 PHE 154 40.623 27.774 20.041 1198CD2 PHE 154 41.705 25.787 19.219 1199 CE1 PHE 154 40.861 27.363 21.3451200 CE2 PHE 154 41.946 25.379 20.523 1201 CZ PHE 154 41.517 26.16321.585 1202 N TYR 155 38.948 27.412 14.696 1203 CA TYR 155 39.098 27.09313.273 1204 C TYR 155 40.399 27.642 12.704 1205 O TYR 155 40.728 28.82312.878 1206 CB TYR 155 37.925 27.670 12.498 1207 CG TYR 155 36.87826.626 12.143 1208 CD1 TYR 155 35.532 26.965 12.087 1209 CD2 TYR 15537.283 25.325 11.871 1210 CE1 TYR 155 34.589 26.000 11.757 1211 CE2 TYR155 36.343 24.360 11.541 1212 CZ TYR 155 34.998 24.701 11.485 1213 OHTYR 155 34.069 23.738 11.166 1214 N ASN 156 41.072 26.784 11.958 1215 CAASN 156 42.381 27.092 11.379 1216 C ASN 156 42.226 27.502 9.928 1217 OASN 156 41.957 26.676 9.053 1218 CB ASN 156 43.217 25.821 11.428 1219 CGASN 156 44.698 26.066 11.163 1220 OD1 ASN 156 45.090 26.814 10.255 1221ND2 ASN 156 45.506 25.410 11.975 1222 N PHE 157 42.505 28.760 9.663 1223CA PHE 157 42.413 29.259 8.294 1224 C PHE 157 43.786 29.501 7.672 1225 OPHE 157 43.881 30.120 6.605 1226 CB PHE 157 41.580 30.534 8.279 1227 CGPHE 157 40.146 30.340 8.764 1228 CD1 PHE 157 39.321 29.410 8.144 1229CD2 PHE 157 39.666 31.095 9.825 1230 CE1 PHE 157 38.016 29.235 8.5861231 CE2 PHE 157 38.361 30.922 10.265 1232 CZ PHE 157 37.536 29.9929.646 1233 N GLY 158 44.832 28.999 8.307 1234 CA GLY 158 46.189 29.2947.843 1235 C GLY 158 46.902 28.091 7.240 1236 O GLY 158 47.685 28.2396.293 1237 N TRP 159 46.672 26.924 7.814 1238 CA TRP 159 47.369 25.7257.348 1239 C TRP 159 46.776 25.224 6.033 1240 O TRP 159 45.568 24.9915.916 1241 CB TRP 159 47.249 24.653 8.424 1242 CG TRP 159 48.486 23.7948.580 1243 CD1 TRP 159 49.470 23.579 7.641 1244 CD2 TRP 159 48.87323.051 9.756 1245 NE1 TRP 159 50.408 22.760 8.179 1246 CE2 TRP 15950.094 22.428 9.445 1247 CE3 TRP 159 48.300 22.881 11.006 1248 CZ2 TRP159 50.731 21.642 10.396 1249 CZ3 TRP 159 48.942 22.092 11.952 1250 CH2TRP 159 50.152 21.475 11.649 1251 N LYS 160 47.624 25.195 5.021 1252 CALYS 160 47.244 24.638 3.723 1253 C LYS 160 47.419 23.122 3.765 1254 OLYS 160 48.291 22.620 4.488 1255 CB LYS 160 48.167 25.248 2.669 1256 CGLYS 160 47.720 24.950 1.238 1257 CD LYS 160 48.755 25.330 0.179 1258 CELYS 160 49.780 24.225 −0.106 1259 NZ LYS 160 50.743 24.013 0.988 1260 NASP 161 46.556 22.404 3.060 1261 CA ASP 161 46.691 20.945 2.953 1262 CASP 161 48.086 20.566 2.463 1263 O ASP 161 48.620 21.144 1.507 1264 CBASP 161 45.627 20.370 2.014 1265 CG ASP 161 45.747 20.900 0.582 1266 OD1ASP 161 45.211 21.967 0.311 1267 OD2 ASP 161 46.358 20.212 −0.223 1268 NTYR 162 48.697 19.685 3.240 1269 CA TYR 162 50.052 19.168 3.003 1270 CTYR 162 51.103 20.280 2.954 1271 O TYR 162 51.941 20.310 2.046 1272 CBTYR 162 50.059 18.374 1.697 1273 CG TYR 162 49.043 17.233 1.651 1274 CD1TYR 162 49.079 16.226 2.609 1275 CD2 TYR 162 48.082 17.204 0.647 1276CE1 TYR 162 48.150 15.194 2.566 1277 CE2 TYR 162 47.153 16.172 0.6031278 CZ TYR 162 47.190 15.170 1.564 1279 OH TYR 162 46.267 14.147 1.5221280 N GLY 163 51.052 21.184 3.919 1281 CA GLY 163 52.056 22.251 3.9881282 C GLY 163 52.548 22.471 5.412 1283 O GLY 163 52.355 21.627 6.2971284 N VAL 164 53.243 23.577 5.615 1285 CA VAL 164 53.727 23.930 6.9571286 C VAL 164 53.446 25.394 7.294 1287 O VAL 164 53.652 26.294 6.4721288 CB VAL 164 55.226 23.647 7.064 1289 CG1 VAL 164 55.522 22.159 7.2351290 CG2 VAL 164 56.004 24.230 5.888 1291 N ALA 165 52.895 25.602 8.4771292 CA ALA 165 52.682 26.960 8.987 1293 C ALA 165 53.917 27.413 9.7631294 O ALA 165 54.829 26.612 10.000 1295 CB ALA 165 51.446 26.974 9.8801296 N SER 166 53.981 28.696 10.081 1297 CA SER 166 55.134 29.222 10.8271298 C SER 166 55.206 28.591 12.210 1299 O SER 166 54.179 28.208 12.7871300 CB SER 166 55.016 30.733 11.008 1301 OG SER 166 54.381 30.97812.260 1302 N LEU 167 56.401 28.624 12.777 1303 CA LEU 167 56.646 28.09114.124 1304 C LEU 167 55.706 28.734 15.133 1305 O LEU 167 54.831 28.05015.675 1306 CB LEU 167 58.081 28.394 14.575 1307 CG LEU 167 59.17327.486 14.000 1308 CD1 LEU 167 59.566 27.847 12.568 1309 CD2 LEU 16760.417 27.574 14.877 1310 N THR 168 55.665 30.056 15.110 1311 CA THR 16854.860 30.796 16.086 1312 C THR 168 53.347 30.667 15.887 1313 O THR 16852.653 30.557 16.905 1314 CB THR 168 55.270 32.261 16.033 1315 OG1 THR168 54.985 32.775 14.736 1316 CG2 THR 168 56.764 32.402 16.293 1317 NTHR 169 52.866 30.447 14.668 1318 CA THR 169 51.425 30.226 14.487 1319 CTHR 169 50.988 28.809 14.853 1320 O THR 169 49.834 28.611 15.248 1321 CBTHR 169 51.041 30.496 13.036 1322 OG1 THR 169 51.736 29.580 12.202 1323CG2 THR 169 51.404 31.909 12.603 1324 N ILE 170 51.907 27.860 14.8711325 CA ILE 170 51.524 26.527 15.321 1326 C ILE 170 51.658 26.442 16.8341327 O ILE 170 50.764 25.907 17.503 1328 CB ILE 170 52.425 25.484 14.6701329 CG1 ILE 170 52.401 25.606 13.152 1330 CG2 ILE 170 51.985 24.08315.080 1331 CD1 ILE 170 53.336 24.592 12.503 1332 N LEU 171 52.59327.206 17.374 1333 CA LEU 171 52.840 27.147 18.816 1334 C LEU 171 51.84027.976 19.612 1335 O LEU 171 51.397 27.517 20.672 1336 CB LEU 171 54.26127.611 19.105 1337 CG LEU 171 55.280 26.719 18.403 1338 CD1 LEU 17156.698 27.206 18.659 1339 CD2 LEU 171 55.126 25.258 18.811 1340 N ASP172 51.303 29.033 19.023 1341 CA ASP 172 50.250 29.760 19.736 1342 C ASP172 48.896 29.081 19.525 1343 O ASP 172 48.082 29.077 20.455 1344 CB ASP172 50.214 31.248 19.350 1345 CG ASP 172 49.557 31.556 18.001 1346 OD1ASP 172 49.834 30.852 17.044 1347 OD2 ASP 172 48.884 32.576 17.929 1348N MET 173 48.792 28.266 18.484 1349 CA MET 173 47.564 27.508 18.254 1350C MET 173 47.456 26.354 19.245 1351 O MET 173 46.462 26.274 19.980 1352CB MET 173 47.595 26.952 16.833 1353 CG MET 173 46.286 26.292 16.3971354 SD MET 173 44.923 27.378 15.898 1355 CE MET 173 44.327 27.97117.498 1356 N VAL 174 48.568 25.667 19.470 1357 CA VAL 174 48.554 24.54120.410 1358 C VAL 174 48.621 24.973 21.875 1359 O VAL 174 48.155 24.21722.733 1360 CB VAL 174 49.698 23.575 20.103 1361 CG1 VAL 174 49.52822.933 18.735 1362 CG2 VAL 174 51.070 24.225 20.219 1363 N LYS 17548.941 26.231 22.137 1364 CA LYS 175 48.966 26.709 23.520 1365 C LYS 17547.573 27.157 23.972 1366 O LYS 175 47.325 27.310 25.173 1367 CB LYS 17549.966 27.858 23.613 1368 CG LYS 175 50.843 27.759 24.861 1369 CD LYS175 50.068 28.004 26.150 1370 CE LYS 175 50.857 27.559 27.372 1371 NZLYS 175 51.160 26.121 27.291 1372 N VAL 176 46.641 27.274 23.040 1373 CAVAL 176 45.268 27.575 23.441 1374 C VAL 176 44.443 26.286 23.556 1375 OVAL 176 43.335 26.301 24.110 1376 CB VAL 176 44.659 28.544 22.428 1377CG1 VAL 176 43.342 29.119 22.938 1378 CG2 VAL 176 45.615 29.697 22.1551379 N MET 177 45.023 25.166 23.148 1380 CA MET 177 44.303 23.887 23.1951381 C MET 177 44.238 23.299 24.602 1382 O MET 177 45.236 22.830 25.1611383 CB MET 177 44.991 22.893 22.268 1384 CG MET 177 44.920 23.35020.817 1385 SD MET 177 45.681 22.240 19.613 1386 CE MET 177 45.30023.164 18.107 1387 N THR 178 43.034 23.311 25.148 1388 CA THR 178 42.78522.687 26.449 1389 C THR 178 42.695 21.175 26.275 1390 O THR 178 41.76920.664 25.637 1391 CB THR 178 41.473 23.232 27.004 1392 OG1 THR 17841.603 24.643 27.118 1393 CG2 THR 178 41.161 22.671 28.388 1394 N PHE179 43.670 20.476 26.832 1395 CA PHE 179 43.723 19.020 26.675 1396 C PHE179 42.899 18.305 27.744 1397 O PHE 179 42.239 17.305 27.441 1398 CB PHE179 45.182 18.599 26.783 1399 CG PHE 179 45.605 17.563 25.750 1400 CD1PHE 179 45.058 17.599 24.474 1401 CD2 PHE 179 46.546 16.595 26.074 1402CE1 PHE 179 45.446 16.663 23.524 1403 CE2 PHE 179 46.933 15.660 25.1241404 CZ PHE 179 46.384 15.693 23.849 1405 N ALA 180 42.832 18.924 28.9171406 CA ALA 180 42.058 18.435 30.076 1407 C ALA 180 42.026 16.914 30.2251408 O ALA 180 41.072 16.267 29.774 1409 CB ALA 180 40.634 18.967 29.9621410 N LEU 181 42.892 16.407 31.091 1411 CA LEU 181 43.068 14.951 31.2661412 C LEU 181 42.018 14.314 32.199 1413 O LEU 181 42.150 13.159 32.6181414 CB LEU 181 44.482 14.717 31.797 1415 CG LEU 181 44.962 13.28431.576 1416 CD1 LEU 181 44.904 12.909 30.099 1417 CD2 LEU 181 46.37013.084 32.128 1418 N GLN 182 41.006 15.088 32.558 1419 CA GLN 182 39.89814.583 33.363 1420 C GLN 182 38.719 14.224 32.460 1421 O GLN 182 37.69713.711 32.931 1422 CB GLN 182 39.495 15.690 34.327 1423 CG GLN 18240.682 16.109 35.188 1424 CD GLN 182 40.349 17.383 35.956 1425 OE1 GLN182 39.583 18.225 35.476 1426 NE2 GLN 182 40.996 17.549 37.096 1427 NGLU 183 38.840 14.551 31.183 1428 CA GLU 183 37.771 14.260 30.223 1429 CGLU 183 38.265 13.284 29.161 1430 O GLU 183 39.333 12.679 29.312 1431 CBGLU 183 37.271 15.551 29.572 1432 CG GLU 183 36.287 16.343 30.444 1433CD GLU 183 36.963 17.180 31.533 1434 OE1 GLU 183 36.262 17.593 32.4441435 OE2 GLU 183 38.143 17.472 31.381 1436 N GLY 184 37.472 13.11128.118 1437 CA GLY 184 37.827 12.176 27.051 1438 C GLY 184 38.821 12.79226.077 1439 O GLY 184 39.341 13.897 26.285 1440 N LYS 185 39.074 12.05225.013 1441 CA LYS 185 40.052 12.451 23.992 1442 C LYS 185 39.645 13.74023.291 1443 O LYS 185 38.475 14.140 23.312 1444 CB LYS 185 40.179 11.36822.916 1445 CG LYS 185 40.876 10.079 23.355 1446 CD LYS 185 39.952 9.03923.985 1447 CE LYS 185 38.873 8.586 23.011 1448 NZ LYS 185 38.014 7.56023.620 1449 N VAL 186 40.632 14.439 22.766 1450 CA VAL 186 40.337 15.58121.896 1451 C VAL 186 40.268 15.048 20.459 1452 O VAL 186 40.880 14.00920.180 1453 CB VAL 186 41.444 16.611 22.095 1454 CG1 VAL 186 42.71416.224 21.343 1455 CG2 VAL 186 40.986 18.009 21.701 1456 N ALA 18739.449 15.633 19.599 1457 CA ALA 187 39.310 15.068 18.244 1458 C ALA 18739.915 15.942 17.149 1459 O ALA 187 39.243 16.836 16.633 1460 CB ALA 18737.830 14.857 17.952 1461 N ILE 188 41.104 15.583 16.700 1462 CA ILE 18841.822 16.361 15.679 1463 C ILE 188 41.389 15.903 14.292 1464 O ILE 18841.661 14.757 13.916 1465 CB ILE 188 43.303 16.068 15.867 1466 CG1 ILE188 43.645 16.132 17.346 1467 CG2 ILE 188 44.165 17.048 15.077 1468 CD1ILE 188 45.105 15.801 17.583 1469 N HIS 189 40.809 16.793 13.511 1470 CAHIS 189 40.123 16.322 12.310 1471 C HIS 189 40.218 17.235 11.076 1472 OHIS 189 40.412 18.452 11.201 1473 CB HIS 189 38.666 16.150 12.734 1474CG HIS 189 37.645 16.907 11.916 1475 ND1 HIS 189 36.854 16.388 10.9621476 CD2 HIS 189 37.343 18.245 12.007 1477 CE1 HIS 189 36.081 17.36610.449 1478 NE2 HIS 189 36.380 18.512 11.098 1479 N CYS 190 40.26716.579 9.918 1480 CA CYS 190 39.981 17.179 8.591 1481 C CYS 190 40.23816.212 7.439 1482 O CYS 190 39.808 15.059 7.500 1483 CB CYS 190 40.65118.521 8.306 1484 SG CYS 190 39.629 19.964 8.719 1485 N HIS 191 41.02416.646 6.462 1486 CA HIS 191 41.066 16.005 5.128 1487 C HIS 191 41.40614.514 5.072 1488 O HIS 191 40.917 13.830 4.165 1489 CB HIS 191 42.09416.759 4.301 1490 CG HIS 191 41.915 16.692 2.796 1491 ND1 HIS 191 40.83716.247 2.123 1492 CD2 HIS 191 42.834 17.096 1.856 1493 CE1 HIS 19141.058 16.361 0.796 1494 NE2 HIS 191 42.293 16.888 0.633 1495 N ALA 19242.169 14.000 6.020 1496 CA ALA 192 42.425 12.564 6.015 1497 C ALA 19242.543 11.995 7.425 1498 O ALA 192 41.792 11.092 7.814 1499 CB ALA 19243.710 12.303 5.240 1500 N GLY 193 43.521 12.502 8.155 1501 CA GLY 19343.855 11.952 9.468 1502 C GLY 193 45.374 11.866 9.584 1503 O GLY 19345.935 11.748 10.677 1504 N LEU 194 46.025 11.919 8.436 1505 CA LEU 19447.486 11.976 8.389 1506 C LEU 194 47.978 13.408 8.189 1507 O LEU 19447.199 14.364 8.323 1508 CB LEU 194 47.962 11.051 7.272 1509 CG LEU 19447.154 11.149 5.974 1510 CD1 LEU 194 47.563 12.342 5.111 1511 CD2 LEU194 47.327 9.872 5.163 1512 N GLY 195 49.288 13.538 8.046 1513 CA GLY195 49.927 14.801 7.650 1514 C GLY 195 49.977 15.824 8.774 1515 O GLY195 50.637 15.643 9.806 1516 N ARG 196 49.081 16.787 8.646 1517 CA ARG196 48.978 17.886 9.603 1518 C ARG 196 48.322 17.443 10.904 1519 O ARG196 48.654 18.000 11.955 1520 CB ARG 196 48.128 18.967 8.956 1521 CG ARG196 48.810 19.526 7.717 1522 CD ARG 196 47.793 20.137 6.766 1523 NE ARG196 46.986 19.058 6.181 1524 CZ ARG 196 45.667 18.928 6.324 1525 NH1 ARG196 44.983 19.818 7.043 1526 NH2 ARG 196 45.036 17.897 5.764 1527 N THR197 47.639 16.307 10.874 1528 CA THR 197 47.051 15.770 12.106 1529 C THR197 48.178 15.219 12.966 1530 O THR 197 48.372 15.661 14.108 1531 CB THR197 46.113 14.611 11.787 1532 OG1 THR 197 45.332 14.898 10.637 1533 CG2THR 197 45.181 14.306 12.952 1534 N GLY 198 49.064 14.504 12.286 1535 CAGLY 198 50.254 13.915 12.899 1536 C GLY 198 51.148 14.995 13.489 1537 OGLY 198 51.301 15.042 14.716 1538 N VAL 199 51.495 15.978 12.671 1539 CAVAL 199 52.389 17.064 13.107 1540 C VAL 199 51.831 17.887 14.267 1541 OVAL 199 52.541 18.074 15.263 1542 CB VAL 199 52.607 18.018 11.935 1543CG1 VAL 199 53.525 19.175 12.315 1544 CG2 VAL 199 53.162 17.298 10.7181545 N LEU 200 50.535 18.156 14.250 1546 CA LEU 200 49.937 19.032 15.2601547 C LEU 200 49.958 18.397 16.646 1548 O LEU 200 50.546 18.976 17.5721549 CB LEU 200 48.501 19.308 14.833 1550 CG LEU 200 47.866 20.42915.646 1551 CD1 LEU 200 48.671 21.714 15.513 1552 CD2 LEU 200 46.43120.658 15.191 1553 N ILE 201 49.574 17.133 16.725 1554 CA ILE 201 49.56516.485 18.038 1555 C ILE 201 50.939 15.940 18.416 1556 O ILE 201 51.25115.910 19.609 1557 CB ILE 201 48.522 15.371 18.065 1558 CG1 ILE 20148.510 14.636 19.395 1559 CG2 ILE 201 48.747 14.392 16.921 1560 CD1 ILE201 47.526 13.469 19.398 1561 N ALA 200 51.836 15.794 17.458 1562 CA ALA200 53.167 15.313 17.798 1563 C ALA 200 54.113 16.437 18.228 1564 O ALA200 55.036 16.173 19.012 1565 CB ALA 200 53.716 14.528 16.615 1566 N CYS203 53.768 17.681 17.924 1567 CA CYS 203 54.506 18.803 18.522 1568 C CYS203 54.186 18.848 20.008 1569 O CYS 203 55.092 18.732 20.846 1570 CB CYS203 54.044 20.150 17.965 1571 SG CYS 203 54.255 20.541 16.215 1572 N TYR204 52.901 18.691 20.289 1573 CA TYR 204 52.381 18.790 21.653 1574 C TYR204 52.749 17.561 22.485 1575 O TYR 204 53.101 17.701 23.662 1576 CB TYR204 50.864 18.893 21.507 1577 CG TYR 204 50.116 19.555 22.660 1578 CD1TYR 204 50.148 20.938 22.789 1579 CD2 TYR 204 49.398 18.789 23.569 1580CE1 TYR 204 49.449 21.556 23.815 1581 CE2 TYR 204 48.698 19.407 24.5961582 CZ TYR 204 48.717 20.790 24.711 1583 OH TYR 204 47.900 21.41525.632 1584 N LEU 205 52.906 16.431 21.818 1585 CA LEU 205 53.356 15.20022.469 1586 C LEU 205 54.809 15.287 22.905 1587 O LEU 205 55.090 15.07824.090 1588 CB LEU 205 53.221 14.057 21.469 1589 CG LEU 205 53.94712.800 21.938 1590 CD1 LEU 205 53.322 12.211 23.199 1591 CD2 LEU 20553.993 11.765 20.824 1592 N VAL 206 55.672 15.817 22.055 1593 CA VAL 20657.088 15.847 22.410 1594 C VAL 206 57.393 16.985 23.378 1595 O VAL 20658.169 16.766 24.318 1596 CB VAL 206 57.900 15.968 21.131 1597 CG1 VAL206 59.388 16.041 21.436 1598 CG2 VAL 206 57.610 14.782 20.220 1599 NPHE 207 56.547 18.003 23.359 1600 CA PHE 207 56.645 19.108 24.318 1601 CPHE 207 56.094 18.744 25.700 1602 O PHE 207 56.551 19.302 26.705 1603 CBPHE 207 55.851 20.278 23.739 1604 CG PHE 207 55.550 21.412 24.716 1605CD1 PHE 207 54.280 21.522 25.271 1606 CD2 PHE 207 56.532 22.338 25.0411607 CE1 PHE 207 53.997 22.549 26.162 1608 CE2 PHE 207 56.247 23.36625.930 1609 CZ PHE 207 54.981 23.470 26.492 1610 N ALA 208 55.203 17.76825.765 1611 CA ALA 208 54.683 17.326 27.059 1612 C ALA 208 55.512 16.17727.620 1613 O ALA 208 55.587 15.994 28.842 1614 CB ALA 208 53.240 16.87026.879 1615 N THR 209 56.200 15.477 26.734 1616 CA THR 209 57.076 14.38827.154 1617 C THR 209 58.424 14.948 27.589 1618 O THR 209 58.584 15.17128.799 1619 CB THR 209 57.205 13.376 26.017 1620 OG1 THR 209 55.89412.910 25.726 1621 CG2 THR 209 58.034 12.158 26.414 1622 N ARG 21059.269 15.261 26.607 1623 CA ARG 210 60.654 15.787 26.732 1624 C ARG 21061.591 15.051 25.762 1625 O ARG 210 62.702 15.514 25.476 1626 CB ARG 21061.214 15.596 28.142 1627 CG ARG 210 62.513 16.344 28.397 1628 CD ARG210 63.009 16.054 29.806 1629 NE ARG 210 63.160 14.602 29.997 1630 CZARG 210 64.308 14.029 30.363 1631 NH1 ARG 210 64.399 12.699 30.423 1632NH2 ARG 210 65.386 14.783 30.593 1633 N MET 211 61.107 13.959 25.1911634 CA MET 211 62.011 13.028 24.496 1635 C MET 211 61.987 13.101 22.9701636 O MET 211 61.642 14.125 22.372 1637 CB MET 211 61.716 11.610 24.9651638 CG MET 211 62.045 11.464 26.447 1639 SD MET 211 61.917 9.793 27.1181640 CE MET 211 63.095 8.963 26.025 1641 N THR 212 62.472 12.025 22.3701642 CA THR 212 62.704 11.973 20.917 1643 C THR 212 61.428 11.990 20.0781644 O THR 212 60.654 11.022 20.036 1645 CB THR 212 63.516 10.724 20.5821646 OG1 THR 212 62.793 9.578 21.008 1647 CG2 THR 212 64.862 10.72321.296 1648 N ALA 213 61.417 12.955 19.174 1649 CA ALA 213 60.283 13.14318.268 1650 C ALA 213 60.223 12.096 17.160 1651 O ALA 213 59.122 11.69016.784 1652 CB ALA 213 60.405 14.527 17.641 1653 N ASP 214 61.346 11.45316.882 1654 CA ASP 214 61.392 10.442 15.820 1655 C ASP 214 60.791 9.11116.267 1656 O ASP 214 60.036 8.495 15.505 1657 CB ASP 214 62.851 10.22115.434 1658 CG ASP 214 63.484 11.524 14.953 1659 OD1 ASP 214 64.10012.192 15.774 1660 OD2 ASP 214 63.304 11.851 13.789 1661 N GLN 21560.890 8.823 17.556 1662 CA GLN 215 60.332 7.569 18.062 1663 C GLN 21558.864 7.766 18.406 1664 O GLN 215 58.042 6.877 18.144 1665 CB GLN 21561.126 7.135 19.285 1666 CG GLN 215 62.579 6.874 18.905 1667 CD GLN 21563.393 6.499 20.138 1668 OE1 GLN 215 62.988 6.770 21.273 1669 NE2 GLN215 64.561 5.930 19.896 1670 N ALA 216 58.519 9.022 18.639 1671 CA ALA216 57.124 9.399 18.840 1672 C ALA 216 56.364 9.429 17.515 1673 O ALA216 55.193 9.035 17.482 1674 CB ALA 216 57.103 10.783 19.475 1675 N ILE217 57.084 9.610 16.419 1676 CA ILE 217 56.467 9.571 15.091 1677 C ILE217 56.199 8.148 14.627 1678 O ILE 217 55.099 7.882 14.127 1679 CB ILE217 57.383 10.289 14.106 1680 CG1 ILE 217 57.254 11.793 14.297 1681 CG2ILE 217 57.084 9.895 12.666 1682 CD1 ILE 217 55.788 12.204 14.249 1683 NILE 218 57.014 7.210 15.082 1684 CA ILE 218 56.752 5.808 14.753 1685 CILE 218 55.648 5.250 15.648 1686 O ILE 218 54.806 4.482 15.170 1687 CBILE 218 58.040 5.015 14.932 1688 CG1 ILE 218 59.150 5.614 14.077 1689CG2 ILE 218 57.828 3.550 14.565 1690 CD1 ILE 218 60.463 4.863 14.2681691 N PHE 219 55.467 5.878 16.799 1692 CA PHE 219 54.383 5.500 17.7071693 C PHE 219 53.045 6.102 17.253 1694 O PHE 219 51.998 5.458 17.3961695 CB PHE 219 54.774 6.017 19.086 1696 CG PHE 219 54.100 5.316 20.2581697 CD1 PHE 219 53.657 4.006 20.127 1698 CD2 PHE 219 53.951 5.98321.466 1699 CE1 PHE 219 53.051 3.369 21.201 1700 CE2 PHE 219 53.3455.346 22.540 1701 CZ PHE 219 52.894 4.040 22.406 1702 N VAL 220 53.1227.177 16.482 1703 CA VAL 220 51.942 7.783 15.849 1704 C VAL 220 51.5177.005 14.602 1705 O VAL 220 50.322 6.913 14.291 1706 CB VAL 220 52.3239.217 15.479 1707 CG1 VAL 220 51.462 9.805 14.369 1708 CG2 VAL 22052.339 10.123 16.705 1709 N ARG 221 52.459 6.272 14.037 1710 CA ARG 22152.173 5.371 12.922 1711 C ARG 221 51.741 3.979 13.380 1712 O ARG 22151.435 3.133 12.530 1713 CB ARG 221 53.448 5.218 12.110 1714 CG ARG 22153.920 6.543 11.536 1715 CD ARG 221 55.208 6.352 10.748 1716 NE ARG 22155.636 7.604 10.111 1717 CZ ARG 221 55.365 7.904 8.839 1718 NH1 ARG 22154.633 7.069 8.098 1719 NH2 ARG 221 55.808 9.047 8.315 1720 N ALA 22251.664 3.754 14.683 1721 CA ALA 222 51.379 2.412 15.198 1722 C ALA 22249.900 2.040 15.278 1723 O ALA 222 49.584 0.854 15.429 1724 CB ALA 22252.005 2.283 16.582 1725 N LYS 223 49.004 3.005 15.150 1726 CA LYS 22347.575 2.667 15.191 1727 C LYS 223 46.885 2.938 13.857 1728 O LYS 22345.687 2.667 13.693 1729 CB LYS 223 46.900 3.472 16.291 1730 CG LYS 22347.630 3.317 17.618 1731 CD LYS 223 46.938 4.095 18.726 1732 CE LYS 22347.809 4.139 19.974 1733 NZ LYS 223 49.073 4.837 19.697 1734 N ARG 22447.666 3.431 12.912 1735 CA ARG 224 47.147 3.846 11.607 1736 C ARG 22448.321 4.292 10.752 1737 O ARG 224 49.103 5.146 11.190 1738 CB ARG 22446.204 5.033 11.801 1739 CG ARG 224 45.387 5.352 10.551 1740 CD ARG 22444.553 6.612 10.753 1741 NE ARG 224 43.516 6.749 9.718 1742 CZ ARG 22442.332 7.317 9.960 1743 NH1 ARG 224 42.083 7.852 11.157 1744 NH2 ARG 22441.408 7.379 9.001 1745 N PRO 225 48.481 3.675 9.594 1746 CA PRO 22549.468 4.142 8.619 1747 C PRO 225 49.187 5.576 8.169 1748 O PRO 22548.168 5.854 7.529 1749 CB PRO 225 49.373 3.178 7.476 1750 CG PRO 22548.264 2.175 7.752 1751 CD PRO 225 47.681 2.550 9.103 1752 N ASN 22650.051 6.484 8.590 1753 CA ASN 226 49.907 7.893 8.213 1754 C ASN 22651.166 8.475 7.572 1755 O ASN 226 51.836 7.819 6.767 1756 CB ASN 22649.475 8.710 9.432 1757 CG ASN 226 50.246 8.368 10.705 1758 OD1 ASN 22651.482 8.407 10.751 1759 ND2 ASN 226 49.477 8.208 11.763 1760 N SER 22751.397 9.745 7.864 1761 CA SER 227 52.518 10.508 7.301 1762 C SER 22752.726 11.761 8.141 1763 O SER 227 51.771 12.228 8.774 1764 CB SER 22752.179 10.912 5.871 1765 OG SER 227 51.010 11.718 5.921 1766 N ILE 22853.915 12.338 8.076 1767 CA ILE 228 54.200 13.523 8.888 1768 C ILE 22855.332 14.377 8.291 1769 O ILE 228 56.360 13.864 7.824 1770 CB ILE 22854.507 13.008 10.301 1771 CG1 ILE 228 54.609 14.098 11.363 1772 CG2 ILE228 55.779 12.168 10.300 1773 CD1 ILE 228 56.030 14.622 11.522 1774 NGLN 229 55.062 15.672 8.204 1775 CA GLN 229 56.072 16.676 7.810 1776 CGLN 229 57.048 16.881 8.970 1777 O GLN 229 56.891 17.816 9.770 1778 CBGLN 229 55.411 18.023 7.490 1779 CG GLN 229 54.343 17.983 6.390 1780 CDGLN 229 52.947 17.774 6.981 1781 OE1 GLN 229 52.486 16.635 7.123 1782NE2 GLN 229 52.279 18.870 7.287 1783 N THR 230 58.161 16.171 8.910 1784CA THR 230 59.014 16.015 10.092 1785 C THR 230 59.873 17.229 10.407 1786O THR 230 59.963 17.584 11.587 1787 CB THR 230 59.896 14.792 9.874 1788OG1 THR 230 59.044 13.696 9.563 1789 CG2 THR 230 60.705 14.438 11.1191790 N ARG 231 60.223 18.023 9.408 1791 CA ARG 231 61.030 19.217 9.7001792 C ARG 231 60.161 20.356 10.238 1793 O ARG 231 60.612 21.111 11.1111794 CB ARG 231 61.757 19.675 8.444 1795 CG ARG 231 62.753 20.779 8.7861796 CD ARG 231 63.495 21.289 7.557 1797 NE ARG 231 64.480 22.312 7.9431798 CZ ARG 231 64.289 23.622 7.765 1799 NH1 ARG 231 63.177 24.062 7.1711800 NH2 ARG 231 65.223 24.491 8.157 1801 N GLY 232 58.870 20.269 9.9541802 CA GLY 232 57.906 21.230 10.483 1803 C GLY 232 57.764 20.996 11.9791804 O GLY 232 58.006 21.913 12.774 1805 N GLN 233 57.638 19.730 12.3461806 CA GLN 233 57.517 19.368 13.759 1807 C GLN 233 58.840 19.487 14.5251808 O GLN 233 58.814 19.807 15.720 1809 CB GLN 233 57.028 17.933 13.8481810 CG GLN 233 56.808 17.533 15.300 1811 CD GLN 233 56.588 16.03615.379 1812 OE1 GLN 233 55.971 15.445 14.486 1813 NE2 GLN 233 57.07315.444 16.456 1814 N LEU 234 59.965 19.441 13.831 1815 CA LEU 234 61.25119.663 14.498 1816 C LEU 234 61.388 21.116 14.934 1817 O LEU 234 61.56121.367 16.133 1818 CB LEU 234 62.386 19.307 13.549 1819 CG LEU 23462.499 17.804 13.337 1820 CD1 LEU 234 63.564 17.482 12.295 1821 CD2 LEU234 62.791 17.087 14.651 1822 N LEU 235 61.009 22.030 14.055 1823 CA LEU235 61.052 23.462 14.382 1824 C LEU 235 59.991 23.830 15.420 1825 O LEU235 60.292 24.524 16.403 1826 CB LEU 235 60.771 24.230 13.096 1827 CGLEU 235 61.899 24.100 12.080 1828 CD1 LEU 235 61.433 24.513 10.689 1829CD2 LEU 235 63.115 24.912 12.510 1830 N CYS 236 58.863 23.145 15.3211831 CA CYS 236 57.731 23.292 16.241 1832 C CYS 236 58.148 22.984 17.6761833 O CYS 236 58.174 23.884 18.528 1834 CB CYS 236 56.717 22.245 15.7841835 SG CYS 236 54.977 22.428 16.227 1836 N VAL 237 58.724 21.807 17.8561837 CA VAL 237 59.107 21.361 19.193 1838 C VAL 237 60.335 22.087 19.7301839 O VAL 237 60.288 22.536 20.881 1840 CB VAL 237 59.393 19.866 19.1381841 CG1 VAL 237 59.988 19.373 20.451 1842 CG2 VAL 237 58.137 19.07818.788 1843 N ARG 238 61.260 22.468 18.863 1844 CA ARG 238 62.478 23.11419.358 1845 C ARG 238 62.259 24.558 19.797 1846 O ARG 238 62.926 24.99520.743 1847 CB ARG 238 63.563 23.055 18.291 1848 CG ARG 238 64.03621.621 18.081 1849 CD ARG 238 65.218 21.556 17.123 1850 NE ARG 23864.868 22.091 15.798 1851 CZ ARG 238 65.183 21.462 14.665 1852 NH1 ARG238 64.849 21.996 13.489 1853 NH2 ARG 238 65.833 20.297 14.707 1854 NGLU 239 61.224 25.211 19.294 1855 CA GLU 239 60.937 26.558 19.784 1856 CGLU 239 59.984 26.516 20.979 1857 O GLU 239 60.149 27.300 21.923 1858 CBGLU 239 60.317 27.376 18.658 1859 CG GLU 239 60.272 28.867 18.988 1860CD GLU 239 61.632 29.523 18.734 1861 OE1 GLU 239 62.520 28.794 18.3061862 OE2 GLU 239 61.628 30.739 18.600 1863 N PHE 240 59.178 25.47021.061 1864 CA PHE 240 58.205 25.384 22.155 1865 C PHE 240 58.848 24.84623.432 1866 O PHE 240 58.475 25.258 24.537 1867 CB PHE 240 57.067 24.47421.715 1868 CG PHE 240 55.720 24.821 22.340 1869 CD1 PHE 240 55.52326.072 22.912 1870 CD2 PHE 240 54.683 23.898 22.316 1871 CE1 PHE 24054.294 26.393 23.474 1872 CE2 PHE 240 53.455 24.219 22.878 1873 CZ PHE240 53.261 25.466 23.459 1874 N THR 241 59.968 24.162 23.268 1875 CA THR241 60.747 23.709 24.427 1876 C THR 241 61.567 24.827 25.066 1877 O THR241 61.885 24.710 26.253 1878 CB THR 241 61.685 22.575 24.028 1879 OG1THR 241 62.454 22.992 22.905 1880 CG2 THR 241 60.919 21.311 23.655 1881N GLN 242 61.686 25.969 24.406 1882 CA GLN 242 62.369 27.114 25.016 1883C GLN 242 61.419 27.821 25.981 1884 O GLN 242 61.806 28.164 27.108 1885CB GLN 242 62.748 28.062 23.892 1886 CG GLN 242 63.592 27.365 22.8371887 CD GLN 242 63.619 28.214 21.573 1888 OE1 GLN 242 62.789 29.11621.400 1889 NE2 GLN 242 64.482 27.833 20.649 1890 N PHE 243 60.14127.719 25.648 1891 CA PHE 243 59.070 28.208 26.515 1892 C PHE 243 58.90527.312 27.737 1893 O PHE 243 58.722 27.804 28.858 1894 CB PHE 243 57.78128.203 25.694 1895 CG PHE 243 56.495 28.262 26.512 1896 CD1 PHE 24355.812 27.090 26.815 1897 CD2 PHE 243 55.996 29.482 26.939 1898 CE1 PHE243 54.649 27.137 27.570 1899 CE2 PHE 243 54.830 29.530 27.692 1900 CZPHE 243 54.160 28.358 28.013 1901 N LEU 244 59.192 26.035 27.547 1902 CALEU 244 59.078 25.073 28.637 1903 C LEU 244 60.281 25.136 29.575 1904 OLEU 244 60.124 24.955 30.788 1905 CB LEU 244 59.005 23.685 28.010 1906CG LEU 244 58.760 22.589 29.040 1907 CD1 LEU 244 57.442 22.817 29.7731908 CD2 LEU 244 58.772 21.215 28.380 1909 N THR 245 61.445 25.48329.052 1910 CA THR 245 62.631 25.506 29.913 1911 C THR 245 63.461 26.79029.845 1912 O THR 245 64.191 27.044 28.880 1913 CB THR 245 63.536 24.31829.576 1914 OG1 THR 245 63.864 24.360 28.194 1915 CG2 THR 245 62.87622.973 29.859 1916 N PRO 246 63.363 27.578 30.903 1917 CA PRO 246 62.13027.756 31.682 1918 C PRO 246 61.264 28.938 31.202 1919 O PRO 246 60.51229.493 32.015 1920 CB PRO 246 62.677 28.110 33.029 1921 CG PRO 24664.034 28.776 32.803 1922 CD PRO 246 64.353 28.571 31.325 1923 N LEU 24761.440 29.408 29.975 1924 CA LEU 247 60.953 30.753 29.656 1925 C LEU 24759.526 30.783 29.120 1926 O LEU 247 59.300 30.898 27.907 1927 CB LEU 24761.916 31.394 28.667 1928 CG LEU 247 61.737 32.908 28.636 1929 CD1 LEU247 61.841 33.491 30.040 1930 CD2 LEU 247 62.756 33.559 27.710 1931 NARG 248 58.631 31.077 30.051 1932 CA ARG 248 57.195 31.190 29.765 1933 CARG 248 56.831 32.488 29.030 1934 O ARG 248 55.842 32.521 28.282 1935 CBARG 248 56.460 31.117 31.100 1936 CG ARG 248 54.948 31.075 30.919 1937CD ARG 248 54.231 30.896 32.252 1938 NE ARG 248 52.782 30.741 32.0461939 CZ ARG 248 52.157 29.563 32.113 1940 NH1 ARG 248 52.846 28.45432.392 1941 NH2 ARG 248 50.839 29.494 31.909 1942 N ASN 249 57.79233.399 28.964 1943 CA ASN 249 57.619 34.659 28.233 1944 C ASN 249 57.75434.469 26.719 1945 O ASN 249 57.318 35.338 25.955 1946 CB ASN 249 58.68235.655 28.690 1947 CG ASN 249 58.612 35.938 30.192 1948 OD1 ASN 24957.616 35.656 30.868 1949 ND2 ASN 249 59.692 36.509 30.697 1950 N ILE250 58.147 33.279 26.287 1951 CA ILE 250 58.235 33.000 24.855 1952 C ILE250 56.867 32.748 24.218 1953 O ILE 250 56.710 33.081 23.039 1954 CB ILE250 59.194 31.832 24.650 1955 CG1 ILE 250 60.592 32.291 25.035 1956 CG2ILE 250 59.185 31.311 23.217 1957 CD1 ILE 250 61.640 31.230 24.746 1958N PHE 251 55.832 32.538 25.020 1959 CA PHE 251 54.483 32.489 24.441 1960C PHE 251 53.999 33.904 24.120 1961 O PHE 251 53.466 34.132 23.027 1962CB PHE 251 53.523 31.842 25.429 1963 CG PHE 251 52.080 31.775 24.9371964 CD1 PHE 251 51.804 31.378 23.634 1965 CD2 PHE 251 51.042 32.10725.796 1966 CE1 PHE 251 50.489 31.326 23.189 1967 CE2 PHE 251 49.72732.053 25.351 1968 CZ PHE 251 49.451 31.665 24.047 1969 N SER 252 54.53034.859 24.867 1970 CA SER 252 54.213 36.271 24.657 1971 C SER 252 55.08436.888 23.563 1972 O SER 252 54.852 38.034 23.168 1973 CB SER 252 54.44137.012 25.967 1974 OG SER 252 53.641 36.382 26.959 1975 N CYS 253 56.05536.133 23.071 1976 CA CYS 253 56.824 36.548 21.899 1977 C CYS 253 56.28835.853 20.649 1978 O CYS 253 56.185 36.474 19.580 1979 CB CYS 253 58.28436.169 22.119 1980 SG CYS 253 59.406 36.564 20.759 1981 N CYS 254 55.74434.662 20.843 1982 CA CYS 254 55.149 33.912 19.732 1983 C CYS 254 53.78934.471 19.348 1984 O CYS 254 53.473 34.505 18.157 1985 CB CYS 254 54.99232.445 20.119 1986 SG CYS 254 56.525 31.502 20.282 1987 N ASP 255 53.10435.098 20.288 1988 CA ASP 255 51.852 35.799 19.963 1989 C ASP 255 52.05536.943 18.944 1990 O ASP 255 51.515 36.807 17.836 1991 CB ASP 255 51.18136.283 21.250 1992 CG ASP 255 50.782 35.099 22.129 1993 OD1 ASP 25550.766 35.269 23.342 1994 OD2 ASP 255 50.430 34.070 21.569 1995 N PRO256 52.873 37.966 19.197 1996 CA PRO 256 53.062 38.993 18.167 1997 C PRO256 53.848 38.544 16.929 1998 O PRO 256 53.637 39.152 15.877 1999 CB PRO256 53.771 40.125 18.844 2000 CG PRO 256 54.195 39.688 20.231 2001 CDPRO 256 53.635 38.290 20.410 2002 N LYS 257 54.577 37.436 16.983 2003 CALYS 257 55.275 36.925 15.791 2004 C LYS 257 54.396 35.967 14.970 2005 OLYS 257 54.698 35.662 13.810 2006 CB LYS 257 56.554 36.234 16.264 2007CG LYS 257 57.405 35.682 15.122 2008 CD LYS 257 57.794 36.754 14.1122009 CE LYS 257 58.627 36.170 12.978 2010 NZ LYS 257 59.863 35.56313.498 2011 N ALA 258 53.275 35.561 15.543 2012 CA ALA 258 52.279 34.78714.802 2013 C ALA 258 51.330 35.735 14.091 2014 O ALA 258 50.635 35.34513.143 2015 CB ALA 258 51.490 33.927 15.782 2016 N HIS 259 51.351 36.97914.546 2017 CA HIS 259 50.622 38.067 13.896 2018 C HIS 259 49.127 37.75013.860 2019 O HIS 259 48.573 37.492 12.783 2020 CB HIS 259 51.163 38.21312.478 2021 CG HIS 259 52.589 38.699 12.265 2022 ND1 HIS 259 53.36739.406 13.109 2023 CD2 HIS 259 53.330 38.485 11.128 2024 CE1 HIS 25954.557 39.642 12.519 2025 NE2 HIS 259 54.534 39.070 11.297 2026 N ALA260 48.498 37.865 15.022 2027 CA ALA 260 47.150 37.325 15.256 2028 C ALA260 46.071 38.294 15.757 2029 O ALA 260 45.088 37.825 16.350 2030 CB ALA260 47.297 36.212 16.289 2031 N VAL 261 46.218 39.591 15.536 2032 CA VAL261 45.226 40.568 16.034 2033 C VAL 261 43.856 40.327 15.381 2034 O VAL261 43.785 39.611 14.374 2035 CB VAL 261 45.810 41.973 15.807 2036 CG1VAL 261 44.925 42.963 15.060 2037 CG2 VAL 261 46.330 42.585 17.101 2038N THR 262 42.783 40.756 16.037 2039 CA THR 262 41.399 40.544 15.539 2040C THR 262 41.018 41.377 14.300 2041 O THR 262 40.131 42.238 14.338 2042CB THR 262 40.421 40.849 16.671 2043 OG1 THR 262 40.629 42.189 17.1062044 CG2 THR 262 40.643 39.920 17.860 2045 N LEU 263 41.686 41.07013.201 2046 CA LEU 263 41.479 41.693 11.893 2047 C LEU 263 41.537 40.59010.837 2048 O LEU 263 41.860 39.450 11.181 2049 CB LEU 263 42.596 42.71611.660 2050 CG LEU 263 42.390 44.000 12.459 2051 CD1 LEU 263 43.54244.971 12.228 2052 CD2 LEU 263 41.061 44.659 12.100 2053 N PRO 26441.177 40.885 9.593 2054 CA PRO 264 41.320 39.894 8.510 2055 C PRO 26442.766 39.614 8.063 2056 O PRO 264 42.989 38.701 7.260 2057 CB PRO 26440.538 40.462 7.367 2058 CG PRO 264 40.144 41.894 7.686 2059 CD PRO 26440.603 42.149 9.111 2060 N GLN 265 43.730 40.369 8.565 2061 CA GLN 26545.139 40.102 8.263 2062 C GLN 265 45.712 39.077 9.235 2063 O GLN 26545.468 39.166 10.443 2064 CB GLN 265 45.912 41.390 8.482 2065 CG GLN 26545.307 42.576 7.752 2066 CD GLN 265 45.867 43.836 8.395 2067 OE1 GLN 26545.727 44.023 9.611 2068 NE2 GLN 265 46.496 44.670 7.587 2069 N TYR 26646.477 38.133 8.718 2070 CA TYR 266 47.222 37.226 9.599 2071 C TYR 26648.533 36.814 8.941 2072 O TYR 266 48.561 36.531 7.737 2073 CB TYR 26646.375 35.993 9.892 2074 CG TYR 266 46.846 35.156 11.082 2075 CD1 TYR266 47.914 34.282 10.957 2076 CD2 TYR 266 46.168 35.250 12.293 2077 CE1TYR 266 48.339 33.541 12.052 2078 CE2 TYR 266 46.590 34.511 13.386 2079CZ TYR 266 47.687 33.669 13.268 2080 OH TYR 266 48.270 33.153 14.4062081 N LEU 267 49.608 36.969 9.700 2082 CA LEU 267 50.990 36.553 9.3512083 C LEU 267 51.687 37.319 8.204 2084 O LEU 267 52.908 37.200 8.0562085 CB LEU 267 50.964 35.051 9.053 2086 CG LEU 267 52.348 34.405 9.0712087 CD1 LEU 267 53.051 34.636 10.406 2088 CD2 LEU 267 52.258 32.9138.766 2089 N ILE 268 50.980 38.145 7.452 2090 CA ILE 268 51.649 38.8996.387 2091 C ILE 268 52.075 40.255 6.934 2092 O ILE 268 53.250 40.4817.243 2093 CB ILE 268 50.691 39.048 5.206 2094 CG1 ILE 268 50.062 37.7044.861 2095 CG2 ILE 268 51.408 39.578 3.968 2096 CD1 ILE 268 49.20137.824 3.609 2097 N ARG 269 51.102 41.140 7.063 2098 CA ARG 269 51.31242.431 7.731 2099 C ARG 269 50.438 42.463 8.973 2100 O ARG 269 49.21042.553 8.849 2101 CB ARG 269 50.903 43.596 6.825 2102 CG ARG 269 51.97744.083 5.846 2103 CD ARG 269 52.201 43.159 4.652 2104 NE ARG 269 53.06043.788 3.638 2105 CZ ARG 269 53.875 43.097 2.838 2106 NH1 ARG 269 54.04241.787 3.030 2107 NH2 ARG 269 54.593 43.730 1.908 2108 N GLN 270 51.04442.321 10.140 2109 CA GLN 270 50.231 42.266 11.360 2110 C GLN 270 50.93642.706 12.647 2111 O GLN 270 51.571 43.768 12.687 2112 CB GLN 270 49.67540.859 11.489 2113 CG GLN 270 48.166 40.801 11.297 2114 CD GLN 27047.509 41.516 12.461 2115 OE1 GLN 270 47.850 41.228 13.619 2116 NE2 GLN270 46.723 42.532 12.148 2117 N ARG 271 50.917 41.825 13.640 2118 CA ARG271 51.167 42.164 15.059 2119 C ARG 271 52.516 42.735 15.486 2120 O ARG271 52.563 43.318 16.574 2121 CB ARG 271 50.955 40.928 15.915 2122 CGARG 271 49.598 40.918 16.595 2123 CD ARG 271 49.564 39.849 17.678 2124NE ARG 271 48.287 39.873 18.398 2125 CZ ARG 271 48.175 39.580 19.6932126 NH1 ARG 271 49.260 39.246 20.394 2127 NH2 ARG 271 46.982 39.63520.288 2128 N HIS 272 53.533 42.761 14.646 2129 CA HIS 272 54.746 43.46515.066 2130 C HIS 272 54.623 44.982 14.917 2131 O HIS 272 55.416 45.72115.510 2132 CB HIS 272 55.959 42.920 14.330 2133 CG HIS 272 56.55041.738 15.067 2134 ND1 HIS 272 56.411 41.487 16.382 2135 CD2 HIS 27257.324 40.730 14.546 2136 CE1 HIS 272 57.072 40.357 16.696 2137 NE2 HIS272 57.637 39.890 15.560 2138 N LEU 273 53.587 45.441 14.231 2139 CA LEU273 53.265 46.868 14.245 2140 C LEU 273 51.779 47.090 14.549 2141 O LEU273 51.421 48.002 15.308 2142 CB LEU 273 53.683 47.545 12.929 2143 CGLEU 273 53.058 46.979 11.648 2144 CD1 LEU 273 52.735 48.096 10.663 2145CD2 LEU 273 53.917 45.906 10.976 2146 N LEU 274 50.958 46.128 14.1612147 CA LEU 274 49.504 46.278 14.283 2148 C LEU 274 48.935 45.785 15.6112149 O LEU 274 47.770 46.074 15.906 2150 CB LEU 274 48.821 45.575 13.1172151 CG LEU 274 49.232 46.196 11.783 2152 CD1 LEU 274 48.627 45.43810.611 2153 CD2 LEU 274 48.849 47.669 11.706 2154 N HIS 275 49.76545.207 16.465 2155 CA HIS 275 49.303 44.912 17.822 2156 C HIS 275 49.35246.205 18.621 2157 O HIS 275 48.359 46.573 19.263 2158 CB HIS 275 50.22443.879 18.454 2159 CG HIS 275 49.801 43.393 19.823 2160 ND1 HIS 27548.552 43.380 20.328 2161 CD2 HIS 275 50.630 42.878 20.790 2162 CE1 HIS275 48.583 42.871 21.576 2163 NE2 HIS 275 49.868 42.560 21.861 2164 NGLY 276 50.365 47.002 18.312 2165 CA GLY 276 50.497 48.347 18.873 2166 CGLY 276 49.388 49.236 18.328 2167 O GLY 276 48.648 49.843 19.109 2168 NTYR 277 49.162 49.149 17.027 2169 CA TYR 277 48.084 49.907 16.380 2170 CTYR 277 46.689 49.582 16.920 2171 O TYR 277 45.977 50.518 17.301 2172 CBTYR 277 48.142 49.604 14.889 2173 CG TYR 277 46.984 50.167 14.072 2174CD1 TYR 277 46.087 49.298 13.462 2175 CD2 TYR 277 46.831 51.540 13.9282176 CE1 TYR 277 45.029 49.801 12.718 2177 CE2 TYR 277 45.773 52.04513.185 2178 CZ TYR 277 44.874 51.174 12.584 2179 OH TYR 277 43.80451.675 11.875 2180 N GLU 278 46.390 48.317 17.176 2181 CA GLU 278 45.06247.975 17.700 2182 C GLU 278 44.933 48.316 19.184 2183 O GLU 278 43.86148.770 19.609 2184 CB GLU 278 44.793 46.490 17.475 2185 CG GLU 27843.383 46.111 17.924 2186 CD GLU 278 43.087 44.647 17.608 2187 OE1 GLU278 43.545 43.794 18.358 2188 OE2 GLU 278 42.500 44.392 16.568 2189 NALA 279 46.056 48.371 19.882 2190 CA ALA 279 46.037 48.798 21.278 2191 CALA 279 45.847 50.308 21.379 2192 O ALA 279 45.014 50.743 22.179 2193 CBALA 279 47.351 48.398 21.939 2194 N ARG 280 46.353 51.048 20.403 2195 CAARG 280 46.166 52.503 20.384 2196 C ARG 280 44.787 52.895 19.860 2197 OARG 280 44.251 53.932 20.276 2198 CB ARG 280 47.246 53.137 19.517 2199CG ARG 280 48.632 52.850 20.080 2200 CD ARG 280 49.720 53.554 19.2782201 NE ARG 280 49.675 53.180 17.854 2202 CZ ARG 280 50.687 52.57817.225 2203 NH1 ARG 280 50.643 52.407 15.902 2204 NH2 ARG 280 51.79052.248 17.901 2205 N LEU 281 44.144 51.986 19.142 2206 CA LEU 281 42.74352.185 18.772 2207 C LEU 281 41.882 52.076 20.018 2208 O LEU 281 41.20453.048 20.377 2209 CB LEU 281 42.304 51.115 17.778 2210 CG LEU 28143.038 51.213 16.447 2211 CD1 LEU 281 42.662 50.045 15.543 2212 CD2 LEU281 42.758 52.542 15.753 2213 N LEU 282 42.214 51.094 20.840 2214 CA LEU282 41.483 50.872 22.092 2215 C LEU 282 41.787 51.938 23.146 2216 O LEU282 40.890 52.265 23.927 2217 CB LEU 282 41.852 49.501 22.662 2218 CGLEU 282 40.897 48.370 22.263 2219 CD1 LEU 282 40.911 48.056 20.768 2220CD2 LEU 282 41.220 47.105 23.049 2221 N LYS 283 42.911 52.629 23.0122222 CA LYS 283 43.269 53.704 23.946 2223 C LYS 283 42.569 55.030 23.6612224 O LYS 283 42.520 55.878 24.558 2225 CB LYS 283 44.771 53.941 23.8722226 CG LYS 283 45.564 52.746 24.386 2227 CD LYS 283 47.047 52.90724.074 2228 CE LYS 283 47.829 51.648 24.428 2229 NZ LYS 283 49.23351.770 24.004 2230 N HIS 284 42.012 55.217 22.474 2231 CA HIS 284 41.22956.438 22.258 2232 C HIS 284 39.739 56.119 22.218 2233 O HIS 284 38.89457.020 22.284 2234 CB HIS 284 41.686 57.187 21.006 2235 CG HIS 28441.436 56.528 19.665 2236 ND1 HIS 284 42.300 55.763 18.972 2237 CD2 HIS284 40.290 56.618 18.910 2238 CE1 HIS 284 41.725 55.372 17.820 2239 NE2HIS 284 40.480 55.897 17.783 2240 N VAL 285 39.429 54.835 22.162 2241 CAVAL 285 38.036 54.374 22.231 2242 C VAL 285 37.418 53.980 23.610 2243 OVAL 285 36.182 54.045 23.637 2244 CB VAL 285 37.992 53.180 21.268 2245CG1 VAL 285 36.726 52.339 21.341 2246 CG2 VAL 285 38.228 53.643 19.8352247 N PRO 286 38.098 53.921 24.763 2248 CA PRO 286 37.892 52.728 25.6132249 C PRO 286 36.593 52.685 26.424 2250 O PRO 286 36.247 51.622 26.9492251 CB PRO 286 39.046 52.699 26.566 2252 CG PRO 286 39.856 53.96726.421 2253 CD PRO 286 39.264 54.692 25.236 2254 N LYS 287 35.870 53.78926.517 2255 CA LYS 287 34.638 53.804 27.311 2256 C LYS 287 33.451 54.40026.558 2257 O LYS 287 32.380 54.572 27.153 2258 CB LYS 287 34.877 54.60028.592 2259 CG LYS 287 35.950 53.958 29.470 2260 CD LYS 287 36.16154.682 30.801 2261 CE LYS 287 35.295 54.139 31.941 2262 NZ LYS 28733.860 54.429 31.781 2263 N ILE 288 33.621 54.727 25.288 2264 CA ILE 28832.524 55.399 24.587 2265 C ILE 288 31.492 54.407 24.035 2266 O ILE 28831.795 53.520 23.226 2267 CB ILE 288 33.098 56.320 23.503 2268 CG1 ILE288 32.021 57.241 22.938 2269 CG2 ILE 288 33.775 55.554 22.373 2270 CD1ILE 288 31.480 58.179 24.012 2271 N ILE 289 30.256 54.614 24.464 2272 CAILE 289 29.096 53.831 24.008 2273 C ILE 289 29.032 53.791 22.474 2274 OILE 289 29.660 54.619 21.800 2275 CB ILE 289 27.852 54.489 24.622 2276CG1 ILE 289 26.565 53.695 24.399 2277 CG2 ILE 289 27.691 55.918 24.1142278 CD1 ILE 289 25.353 54.411 24.985 2279 N HIS 290 28.505 52.68121.969 2280 CA HIS 290 28.391 52.354 20.529 2281 C HIS 290 29.670 51.71719.987 2282 O HIS 290 29.666 50.518 19.683 2283 CB HIS 290 28.015 53.56719.675 2284 CG HIS 290 26.680 54.193 20.023 2285 ND1 HIS 290 25.46353.686 19.753 2286 CD2 HIS 290 26.481 55.391 20.668 2287 CE1 HIS 29024.517 54.525 20.221 2288 NE2 HIS 290 25.148 55.579 20.787 2289 N LEU291 30.789 52.412 20.100 2290 CA LEU 291 32.033 51.859 19.564 2291 C LEU291 32.629 50.847 20.540 2292 O LEU 291 32.973 49.733 20.124 2293 CB LEU291 33.004 52.998 19.285 2294 CG LEU 291 34.173 52.519 18.433 2295 CD1LEU 291 33.679 51.767 17.201 2296 CD2 LEU 291 35.061 53.689 18.029 2297N VAL 292 32.408 51.086 21.822 2298 CA VAL 292 32.774 50.101 22.846 2299C VAL 292 31.684 49.050 23.046 2300 O VAL 292 31.981 47.952 23.527 2301CB VAL 292 33.104 50.844 24.135 2302 CG1 VAL 292 33.169 49.957 25.3712303 CG2 VAL 292 34.420 51.570 23.949 2304 N CYS 293 30.541 49.24822.411 2305 CA CYS 293 29.507 48.217 22.453 2306 C CYS 293 29.878 47.12621.455 2307 O CYS 293 29.913 45.945 21.823 2308 CB CYS 293 28.164 48.83322.084 2309 SG CYS 293 26.763 47.692 22.050 2310 N LYS 294 30.471 47.55520.351 2311 CA LYS 294 30.979 46.614 19.352 2312 C LYS 294 32.304 46.01519.810 2313 O LYS 294 32.482 44.793 19.728 2314 CB LYS 294 31.204 47.38418.059 2315 CG LYS 294 29.934 48.104 17.628 2316 CD LYS 294 30.19449.019 16.440 2317 CE LYS 294 28.946 49.812 16.072 2318 NZ LYS 29429.217 50.721 14.947 2319 N LEU 295 33.079 46.808 20.531 2320 CA LEU 29534.361 46.341 21.066 2321 C LEU 295 34.179 45.257 22.127 2322 O LEU 29534.752 44.171 21.973 2323 CB LEU 295 35.077 47.537 21.681 2324 CG LEU295 36.442 47.161 22.244 2325 CD1 LEU 295 37.346 46.611 21.146 2326 CD2LEU 295 37.089 48.363 22.923 2327 N LEU 296 33.212 45.429 23.014 2328 CALEU 296 32.970 44.417 24.046 2329 C LEU 296 32.164 43.239 23.519 2330 OLEU 296 32.369 42.116 23.992 2331 CB LEU 296 32.243 45.050 25.223 2332CG LEU 296 33.133 46.049 25.951 2333 CD1 LEU 296 32.373 46.714 27.0922334 CD2 LEU 296 34.400 45.377 26.471 2335 N LEU 297 31.474 43.42722.406 2336 CA LEU 297 30.824 42.294 21.751 2337 C LEU 297 31.868 41.42621.055 2338 O LEU 297 31.866 40.206 21.252 2339 CB LEU 297 29.826 42.82220.728 2340 CG LEU 297 29.079 41.686 20.039 2341 CD1 LEU 297 28.33140.828 21.055 2342 CD2 LEU 297 28.121 42.228 18.986 2343 N ASP 29832.913 42.063 20.547 2344 CA ASP 298 34.019 41.333 19.921 2345 C ASP 29834.895 40.655 20.971 2346 O ASP 298 35.296 39.505 20.767 2347 CB ASP 29834.881 42.314 19.129 2348 CG ASP 298 34.088 42.994 18.016 2349 OD1 ASP298 33.227 42.337 17.444 2350 OD2 ASP 298 34.453 44.109 17.664 2351 NLEU 299 34.934 41.230 22.164 2352 CA LEU 299 35.699 40.648 23.277 2353 CLEU 299 34.935 39.529 23.991 2354 O LEU 299 35.540 38.725 24.710 2355 CBLEU 299 36.024 41.752 24.284 2356 CG LEU 299 37.442 42.316 24.157 2357CD1 LEU 299 37.735 42.931 22.791 2358 CD2 LEU 299 37.703 43.343 25.2522359 N ALA 300 33.634 39.452 23.757 2360 CA ALA 300 32.828 38.332 24.2522361 C ALA 300 32.707 37.241 23.190 2362 O ALA 300 32.213 36.139 23.4582363 CB ALA 300 31.442 38.849 24.619 2364 N GLU 301 33.155 37.558 21.9892365 CA GLU 301 33.195 36.566 20.927 2366 C GLU 301 34.561 35.897 20.9002367 O GLU 301 34.582 34.684 21.068 2368 CB GLU 301 32.883 37.222 19.5872369 CG GLU 301 31.460 37.771 19.529 2370 CD GLU 301 30.432 36.68119.818 2371 OE1 GLU 301 29.425 37.000 20.433 2372 OE2 GLU 301 30.68835.542 19.449 2373 OXT GLU 301 35.530 36.567 20.587

TABLE IX Atom Atom Residue No name Residue No x coord y coord z coord 1N MET 1 1.491 5.335 9.487 5 CA MET 1 2.465 4.265 9.217 6 CB MET 1 2.3023.734 7.795 7 CG MET 1 0.916 3.146 7.555 8 SD MET 1 0.637 2.487 5.894 9CE MET 1 −1.071 1.927 6.088 10 C MET 1 3.899 4.759 9.385 11 O MET 14.181 5.962 9.368 12 N GLY 2 4.795 3.807 9.565 14 CA GLY 2 6.223 4.1219.650 15 C GLY 2 6.848 3.946 8.275 16 O GLY 2 7.036 2.817 7.808 17 N VAL3 7.195 5.058 7.649 19 CA VAL 3 7.712 4.996 6.279 20 CB VAL 3 7.3076.264 5.538 21 CG1 VAL 3 5.795 6.314 5.406 22 CG2 VAL 3 7.819 7.5256.223 23 C VAL 3 9.223 4.793 6.234 24 O VAL 3 9.760 4.480 5.165 25 N GLN4 9.859 5.000 7.381 27 CA GLN 4 11.277 4.693 7.677 28 CB GLN 4 12.2665.066 6.568 29 CG GLN 4 12.590 3.899 5.628 30 CD GLN 4 13.264 2.7286.347 31 OE1 GLN 4 12.720 2.136 7.287 32 NE2 GLN 4 14.429 2.364 5.841 35C GLN 4 11.684 5.427 8.942 36 O GLN 4 12.123 6.584 8.860 37 N PRO 511.671 4.714 10.060 38 CA PRO 5 11.800 5.329 11.392 39 CB PRO 5 11.8304.177 12.351 40 CG PRO 5 11.564 2.883 11.597 41 CD PRO 5 11.397 3.27610.139 42 C PRO 5 13.051 6.203 11.511 43 O PRO 5 14.083 5.883 10.911 44N PRO 6 12.944 7.340 12.189 45 CA PRO 6 11.729 7.784 12.906 46 CB PRO 612.248 8.754 13.921 47 CG PRO 6 13.660 9.172 13.541 48 CD PRO 6 14.0568.277 12.379 49 C PRO 6 10.660 8.489 12.051 50 O PRO 6 9.716 9.06312.611 51 N ASN 7 10.857 8.539 10.745 53 CA ASN 7 9.926 9.200 9.833 54CB ASN 7 10.606 9.316 8.470 55 CG ASN 7 11.924 10.089 8.588 56 OD1 ASN 711.916 11.300 8.837 57 ND2 ASN 7 13.033 9.386 8.417 60 C ASN 7 8.5978.456 9.689 61 O ASN 7 8.533 7.261 9.358 62 N PHE 8 7.553 9.199 10.02064 CA PHE 8 6.150 8.809 9.821 65 CB PHE 8 5.392 8.898 11.140 66 CG PHE 85.888 7.982 12.252 67 CD1 PHE 8 5.687 6.612 12.162 68 CE1 PHE 8 6.1305.775 13.177 69 CZ PHE 8 6.771 6.309 14.287 70 CE2 PHE 8 6.966 7.68114.379 71 CD2 PHE 8 6.524 8.518 13.364 72 C PHE 8 5.507 9.783 8.837 73 OPHE 8 4.278 9.890 8.725 74 N SER 9 6.363 10.593 8.240 76 CA SER 9 5.93911.629 7.296 77 CB SER 9 7.187 12.341 6.785 78 OG SER 9 7.860 12.8967.909 79 C SER 9 5.149 11.062 6.120 80 O SER 9 5.273 9.875 5.797 81 NTRP 10 4.124 11.825 5.769 83 CA TRP 10 3.278 11.625 4.582 84 CB TRP 104.120 11.113 3.417 85 CG TRP 10 3.363 10.272 2.411 86 CD1 TRP 10 2.27210.620 1.641 87 NE1 TRP 10 1.907 9.534 0.913 89 CE2 TRP 10 2.716 8.4861.157 90 CZ2 TRP 10 2.726 7.175 0.704 91 CH2 TRP 10 3.703 6.302 1.162 92CZ3 TRP 10 4.671 6.721 2.063 93 CE3 TRP 10 4.662 8.032 2.533 94 CD2 TRP10 3.682 8.905 2.087 95 C TRP 10 2.055 10.739 4.804 96 O TRP 10 0.95911.140 4.401 97 N VAL 11 2.199 9.670 5.571 99 CA VAL 11 1.107 8.7045.726 100 CB VAL 11 1.720 7.350 6.036 101 CG1 VAL 11 2.189 6.629 4.780102 CG2 VAL 11 2.857 7.524 7.030 103 C VAL 11 0.112 9.064 6.822 104 OVAL 11 −0.896 8.367 6.979 105 N LEU 12 0.369 10.124 7.569 107 CA LEU 12−0.601 10.515 8.591 108 CB LEU 12 0.091 11.328 9.674 109 CG LEU 12 1.07910.478 10.462 110 CD1 LEU 12 1.786 11.316 11.520 111 CD2 LEU 12 0.3789.287 11.108 112 C LEU 12 −1.777 11.285 7.989 113 O LEU 12 −1.621 12.2837.269 114 N PRO 13 −2.957 10.743 8.241 115 CA PRO 13 −4.199 11.444 7.950116 CB PRO 13 −5.273 10.409 8.098 117 CG PRO 13 −4.680 9.188 8.784 118CD PRO 13 −3.195 9.477 8.939 119 C PRO 13 −4.408 12.575 8.944 120 O PRO13 −3.895 12.543 10.069 121 N GLY 14 −5.316 13.464 8.588 123 CA GLY 14−5.657 14.607 9.443 124 C GLY 14 −6.418 14.212 10.707 125 O GLY 14−6.397 14.939 11.706 126 N ARG 15 −7.001 13.023 10.689 128 CA ARG 15−7.730 12.499 11.846 129 CB ARG 15 −8.661 11.403 11.348 130 CG ARG 15−9.606 11.903 10.265 131 CD ARG 15 −10.433 10.749 9.714 132 NE ARG 15−9.549 9.689 9.203 133 CZ ARG 15 −9.713 8.395 9.487 134 NH1 ARG 15−8.852 7.493 9.009 135 NH2 ARG 15 −10.724 8.004 10.266 136 C ARG 15−6.826 11.893 12.923 137 O ARG 15 −7.320 11.614 14.022 138 N LEU 16−5.536 11.752 12.655 140 CA LEU 16 −4.617 11.203 13.658 141 CB LEU 16−4.132 9.836 13.175 142 CG LEU 16 −3.840 8.860 14.315 143 CD1 LEU 16−2.597 9.224 15.121 144 CD2 LEU 16 −5.051 8.686 15.225 145 C LEU 16−3.444 12.172 13.827 146 O LEU 16 −2.326 11.909 13.361 147 N ALA 17−3.711 13.270 14.517 149 CA ALA 17 −2.708 14.334 14.650 150 CB ALA 17−2.682 15.115 13.345 151 C ALA 17 −2.995 15.299 15.798 152 O ALA 17−4.154 15.507 16.176 153 N GLY 18 −1.933 15.892 16.324 155 CA GLY 18−2.057 16.952 17.334 156 C GLY 18 −1.412 16.613 18.680 157 O GLY 18−1.545 17.402 19.618 158 N LEU 19 −0.564 15.591 18.666 160 CA LEU 190.035 14.921 19.847 161 CB LEU 19 1.544 14.920 19.643 162 CG LEU 192.278 13.938 20.552 163 CD1 LEU 19 3.524 13.459 19.855 164 CD2 LEU 192.635 14.450 21.947 165 C LEU 19 −0.256 15.506 21.230 166 O LEU 19 0.21316.608 21.556 167 N ALA 20 −0.944 14.702 22.032 169 CA ALA 20 −1.16214.939 23.474 170 CB ALA 20 −1.564 16.392 23.784 171 C ALA 20 −2.23114.008 24.032 172 O ALA 20 −2.310 12.826 23.693 173 N LEU 21 −3.10014.615 24.826 175 CA LEU 21 −4.236 13.948 25.491 176 CB LEU 21 −4.83514.975 26.447 177 CG LEU 21 −3.851 15.345 27.552 178 CD1 LEU 21 −4.42016.451 28.432 179 CD2 LEU 21 −3.485 14.119 28.391 180 C LEU 21 −5.29413.449 24.494 181 O LEU 21 −5.094 13.581 23.281 182 N PRO 22 −6.30012.717 24.959 183 CA PRO 22 −7.377 12.247 24.063 184 CB PRO 22 −7.89611.023 24.748 185 CG PRO 22 −7.465 11.055 26.210 186 CD PRO 22 −6.50512.227 26.331 187 C PRO 22 −8.553 13.223 23.820 188 O PRO 22 −9.52912.809 23.185 189 N ARG 23 −8.486 14.464 24.285 191 CA ARG 23 −9.66715.352 24.237 192 CB ARG 23 −10.046 15.734 25.662 193 CG ARG 23 −10.45614.530 26.501 194 CD ARG 23 −10.859 14.964 27.907 195 NE ARG 23 −11.98515.913 27.853 196 CZ ARG 23 −12.838 16.111 28.862 197 NH1 ARG 23 −12.71415.413 29.993 198 NH2 ARG 23 −13.836 16.986 28.726 199 C ARG 23 −9.45016.656 23.456 200 O ARG 23 −9.149 17.686 24.069 201 N LEU 24 −9.66516.608 22.147 203 CA LEU 24 −9.565 17.774 21.233 204 CB LEU 24 −8.12718.307 21.278 205 CG LEU 24 −8.022 19.818 21.495 206 CD1 LEU 24 −9.00620.340 22.532 207 CD2 LEU 24 −6.596 20.227 21.837 208 C LEU 24 −10.00317.234 19.852 209 O LEU 24 −10.485 16.097 19.870 210 N PRO 25 −9.99517.987 18.748 211 CA PRO 25 −10.621 17.497 17.498 212 CB PRO 25 −10.37118.560 16.469 213 CG PRO 25 −9.727 19.760 17.133 214 CD PRO 25 −9.58319.392 18.599 215 C PRO 25 −10.094 16.145 17.008 216 O PRO 25 −10.70415.105 17.284 217 N ALA 26 −8.992 16.170 16.273 219 CA ALA 26 −8.38814.935 15.754 220 CB ALA 26 −7.240 15.296 14.821 221 C ALA 26 −7.86914.076 16.898 222 O ALA 26 −7.708 14.561 18.027 223 N HIS 27 −7.64612.801 16.628 225 CA HIS 27 −7.168 11.940 17.701 226 CB HIS 27 −7.67810.528 17.541 227 CG HIS 27 −7.659 9.815 18.875 228 ND1 HIS 27 −7.7258.493 19.089 230 CE1 HIS 27 −7.678 8.251 20.413 231 NE2 HIS 27 −7.5879.443 21.046 232 CD2 HIS 27 −7.580 10.418 20.109 233 C HIS 27 −5.65011.995 17.761 234 O HIS 27 −4.904 11.498 16.911 235 N TYR 28 −5.21712.620 18.834 237 CA TYR 28 −3.826 12.997 19.016 238 CB TYR 28 −3.80414.505 19.215 239 CG TYR 28 −4.532 15.170 20.393 240 CD1 TYR 28 −3.83316.119 21.131 241 CE1 TYR 28 −4.425 16.747 22.214 242 CZ TYR 28 −5.74016.451 22.543 243 OH TYR 28 −6.264 16.927 23.728 244 CE2 TYR 28 −6.46615.559 21.768 245 CD2 TYR 28 −5.868 14.931 20.690 246 C TYR 28 −3.11012.230 20.131 247 O TYR 28 −1.874 12.301 20.225 248 N GLN 29 −3.83911.342 20.788 250 CA GLN 29 −3.247 10.517 21.843 251 CB GLN 29 −4.3859.917 22.672 252 CG GLN 29 −3.869 9.030 23.803 253 CD GLN 29 −3.1439.854 24.860 254 OE1 GLN 29 −3.757 10.673 25.555 255 NE2 GLN 29 −1.8719.544 25.050 258 C GLN 29 −2.375 9.394 21.285 259 O GLN 29 −1.329 9.08821.874 260 N PHE 30 −2.619 9.006 20.041 262 CA PHE 30 −1.791 7.95219.436 263 CB PHE 30 −2.497 7.342 18.237 264 CG PHE 30 −3.503 6.26118.605 265 CD1 PHE 30 −3.079 5.135 19.297 266 CE1 PHE 30 −3.990 4.14419.638 267 CZ PHE 30 −5.326 4.279 19.282 268 CE2 PHE 30 −5.749 5.40318.584 269 CD2 PHE 30 −4.837 6.395 18.244 270 C PHE 30 −0.414 8.44719.020 271 O PHE 30 0.547 7.686 19.186 272 N LEU 31 −0.264 9.749 18.837274 CA LEU 31 1.061 10.274 18.516 275 CB LEU 31 0.934 11.609 17.795 276CG LEU 31 0.348 11.458 16.398 277 CD1 LEU 31 0.303 12.815 15.710 278 CD2LEU 31 1.167 10.478 15.563 279 C LEU 31 1.892 10.448 19.782 280 O LEU 313.110 10.218 19.744 281 N LEU 32 1.213 10.543 20.917 283 CA LEU 32 1.91210.610 22.202 284 CB LEU 32 0.954 11.175 23.258 285 CG LEU 32 1.61211.511 24.603 286 CD1 LEU 32 0.816 12.572 25.350 287 CD2 LEU 32 1.83310.299 25.506 288 C LEU 32 2.376 9.206 22.566 289 O LEU 32 3.512 9.03723.018 290 N ASP 33 1.651 8.222 22.058 292 CA ASP 33 2.010 6.818 22.275293 CB ASP 33 0.774 5.950 22.040 294 CG ASP 33 −0.431 6.392 22.875 295OD1 ASP 33 −0.238 6.939 23.956 296 OD2 ASP 33 −1.543 6.171 22.412 297 CASP 33 3.118 6.362 21.315 298 O ASP 33 3.711 5.298 21.525 299 N LEU 343.419 7.166 20.305 301 CA LEU 34 4.511 6.851 19.378 302 CB LEU 34 4.0527.158 17.956 303 CG LEU 34 2.922 6.235 17.514 304 CD1 LEU 34 2.354 6.67216.169 305 CD2 LEU 34 3.385 4.782 17.460 306 C LEU 34 5.785 7.647 19.675307 O LEU 34 6.847 7.328 19.125 308 N GLY 35 5.682 8.668 20.512 310 CAGLY 35 6.859 9.465 20.889 311 C GLY 35 7.265 10.450 19.793 312 O GLY 358.458 10.628 19.505 313 N VAL 36 6.268 11.034 19.151 315 CA VAL 36 6.52112.009 18.082 316 CB VAL 36 5.254 12.078 17.224 317 CG1 VAL 36 5.33113.109 16.106 318 CG2 VAL 36 4.917 10.708 16.646 319 C VAL 36 6.89113.362 18.699 320 O VAL 36 6.422 13.691 19.792 321 N ARG 37 7.864 14.04218.120 323 CA ARG 37 8.238 15.361 18.639 324 CB ARG 37 9.693 15.32919.087 325 CG ARG 37 9.893 14.311 20.204 326 CD ARG 37 9.104 14.67821.455 327 NE ARG 37 9.179 13.596 22.448 328 CZ ARG 37 8.463 13.59123.575 329 NH1 ARG 37 7.657 14.618 23.857 330 NH2 ARG 37 8.571 12.57124.429 331 C ARG 37 8.034 16.436 17.584 332 O ARG 37 7.786 17.608 17.905333 N HIS 38 8.139 16.027 16.332 335 CA HIS 38 7.888 16.951 15.219 336CB HIS 38 9.023 16.869 14.206 337 CG HIS 38 10.205 17.779 14.482 338 ND1HIS 38 10.966 17.831 15.593 340 CE1 HIS 38 11.916 18.774 15.440 341 NE2HIS 38 11.751 19.325 14.216 342 CD2 HIS 38 10.702 18.721 13.615 343 CHIS 38 6.569 16.626 14.536 344 O HIS 38 6.286 15.454 14.257 345 N LEU 395.793 17.660 14.264 347 CA LEU 39 4.482 17.485 13.632 348 CB LEU 393.408 17.536 14.710 349 CG LEU 39 2.030 17.272 14.114 350 CD1 LEU 391.905 15.823 13.654 351 CD2 LEU 39 0.936 17.606 15.114 352 C LEU 394.203 18.580 12.602 353 O LEU 39 3.881 19.719 12.956 354 N VAL 40 4.30218.235 11.332 356 CA VAL 40 4.016 19.234 10.292 357 CB VAL 40 5.12319.172 9.240 358 CG1 VAL 40 4.983 20.265 8.183 359 CG2 VAL 40 6.49219.273 9.902 360 C VAL 40 2.627 19.002 9.684 361 O VAL 40 2.229 17.8569.445 362 N SER 41 1.867 20.078 9.562 364 CA SER 41 0.526 20.034 8.961365 CB SER 41 −0.424 20.839 9.847 366 OG SER 41 −1.720 20.853 9.249 367C SER 41 0.551 20.681 7.584 368 O SER 41 0.581 21.912 7.507 369 N LEU 420.423 19.890 6.530 371 CA LEU 42 0.473 20.457 5.169 372 CB LEU 42 1.04019.439 4.187 373 CG LEU 42 2.561 19.439 4.205 374 CD1 LEU 42 3.09818.599 3.056 375 CD2 LEU 42 3.089 20.859 4.070 376 C LEU 42 −0.86520.942 4.621 377 O LEU 42 −0.890 21.612 3.582 378 N THR 43 −1.949 20.6685.323 380 CA THR 43 −3.266 21.116 4.856 381 CB THR 43 −4.205 19.9194.924 382 OG1 THR 43 −3.537 18.825 4.317 383 CG2 THR 43 −5.511 20.1514.171 384 C THR 43 −3.789 22.276 5.708 385 O THR 43 −4.857 22.835 5.428386 N GLU 44 −2.938 22.735 6.612 388 CA GLU 44 −3.326 23.674 7.668 389CB GLU 44 −3.472 25.086 7.107 390 CG GLU 44 −3.808 26.101 8.198 391 CDGLU 44 −2.818 25.947 9.340 392 OE1 GLU 44 −1.659 26.254 9.101 393 OE2GLU 44 −3.167 25.267 10.304 394 C GLU 44 −4.610 23.234 8.359 395 O GLU44 −5.693 23.788 8.142 396 N ARG 45 −4.467 22.225 9.195 398 CA ARG 45−5.610 21.795 10.000 399 CB ARG 45 −6.112 20.443 9.512 400 CG ARG 45−4.998 19.413 9.440 401 CD ARG 45 −5.533 18.082 8.933 402 NE ARG 45−6.158 18.243 7.613 403 CZ ARG 45 −7.360 17.748 7.307 404 NH1 ARG 45−7.936 18.074 6.149 405 NH2 ARG 45 −8.042 17.044 8.214 406 C ARG 45−5.271 21.765 11.484 407 O ARG 45 −5.920 21.050 12.257 408 N GLY 46−4.287 22.556 11.883 410 CA GLY 46 −3.879 22.543 13.291 411 C GLY 46−2.936 23.682 13.673 412 O GLY 46 −1.737 23.644 13.379 413 N PRO 47−3.489 24.660 14.373 414 CA PRO 47 −2.680 25.637 15.110 415 CB PRO 47−3.673 26.591 15.703 416 CG PRO 47 −5.080 26.075 15.456 417 CD PRO 47−4.919 24.787 14.669 418 C PRO 47 −1.843 24.947 16.187 419 O PRO 47−2.299 23.969 16.796 420 N PRO 48 −0.721 25.557 16.550 421 CA PRO 480.272 24.897 17.414 422 CB PRO 48 1.522 25.708 17.256 423 CG PRO 481.197 26.987 16.502 424 CD PRO 48 −0.262 26.872 16.096 425 C PRO 48−0.127 24.827 18.891 426 O PRO 48 0.454 24.032 19.638 427 N HIS 49−1.260 25.425 19.224 429 CA HIS 49 −1.739 25.515 20.602 430 CB HIS 49−2.794 26.615 20.655 431 CG HIS 49 −2.372 27.894 19.956 432 ND1 HIS 49−1.423 28.759 20.362 434 CE1 HIS 49 −1.329 29.769 19.473 435 NE2 HIS 49−2.234 29.539 18.494 436 CD2 HIS 49 −2.886 28.389 18.780 437 C HIS 49−2.352 24.199 21.072 438 O HIS 49 −2.217 23.859 22.254 439 N SER 50−2.702 23.347 20.119 441 CA SER 50 −3.216 22.015 20.457 442 CB SER 50−3.907 21.431 19.225 443 OG SER 50 −2.950 21.313 18.179 444 C SER 50−2.084 21.083 20.903 445 O SER 50 −2.283 20.257 21.799 446 N ASP 51−0.872 21.410 20.479 448 CA ASP 51 0.321 20.654 20.852 449 CB ASP 511.157 20.376 19.601 450 CG ASP 51 0.496 19.364 18.665 451 OD1 ASP 51−0.494 19.737 18.049 452 OD2 ASP 51 1.186 18.410 18.320 453 C ASP 511.168 21.440 21.843 454 O ASP 51 2.274 21.009 22.193 455 N SER 52 0.63422.550 22.336 457 CA SER 52 1.413 23.466 23.179 458 CB SER 52 0.80224.858 23.083 459 OG SER 52 1.568 25.732 23.898 460 C SER 52 1.47023.023 24.641 461 O SER 52 2.359 23.465 25.382 462 N CYS 53 0.635 22.06725.019 464 CA CYS 53 0.740 21.518 26.376 465 CB CYS 53 −0.570 20.83526.765 466 SG CYS 53 −2.008 21.929 26.788 467 C CYS 53 1.979 20.61126.536 468 O CYS 53 2.767 20.904 27.440 469 N PRO 54 2.189 19.558 25.743470 CA PRO 54 3.504 18.890 25.774 471 CB PRO 54 3.262 17.546 25.163 472CG PRO 54 1.899 17.549 24.486 473 CD PRO 54 1.299 18.920 24.756 474 CPRO 54 4.637 19.621 25.023 475 O PRO 54 5.793 19.194 25.124 476 N GLY 554.325 20.660 24.261 478 CA GLY 55 5.357 21.460 23.588 479 C GLY 55 5.92520.770 22.348 480 O GLY 55 7.122 20.465 22.285 481 N LEU 56 5.065 20.51021.379 483 CA LEU 56 5.513 19.864 20.131 484 CB LEU 56 4.428 19.01319.467 485 CG LEU 56 4.179 17.665 20.127 486 CD1 LEU 56 5.480 16.99420.538 487 CD2 LEU 56 3.241 17.785 21.326 488 C LEU 56 5.967 20.88419.104 489 O LEU 56 5.544 22.047 19.114 490 N THR 57 6.706 20.384 18.131492 CA THR 57 7.177 21.224 17.028 493 CB THR 57 8.523 20.687 16.562 494OG1 THR 57 9.308 20.384 17.709 495 CG2 THR 57 9.268 21.705 15.708 496 CTHR 57 6.183 21.179 15.871 497 O THR 57 6.384 20.437 14.901 498 N LEU 585.091 21.913 16.020 500 CA LEU 58 4.046 21.941 14.992 501 CB LEU 582.690 22.178 15.663 502 CG LEU 58 1.500 21.585 14.893 503 CD1 LEU 580.230 21.637 15.731 504 CD2 LEU 58 1.235 22.237 13.539 505 C LEU 584.343 23.041 13.977 506 O LEU 58 4.370 24.233 14.307 507 N HIS 59 4.57422.631 12.743 509 CA HIS 59 4.785 23.612 11.675 510 CB HIS 59 6.13723.380 11.021 511 CG HIS 59 7.277 23.803 11.928 512 ND1 HIS 59 7.25224.797 12.839 514 CE1 HIS 59 8.452 24.877 13.445 515 NE2 HIS 59 9.24223.914 12.919 516 CD2 HIS 59 8.529 23.240 11.990 517 C HIS 59 3.63023.602 10.680 518 O HIS 59 3.236 22.562 10.138 519 N ARG 60 3.090 24.79010.472 521 CA ARG 60 1.839 24.962 9.727 522 CB ARG 60 1.040 26.00810.485 523 CG ARG 60 0.871 25.621 11.944 524 CD ARG 60 0.279 26.77612.733 525 NE ARG 60 −1.075 27.109 12.272 526 CZ ARG 60 −1.714 28.22512.627 527 NH1 ARG 60 −1.103 29.125 13.399 528 NH2 ARG 60 −2.954 28.45212.192 529 C ARG 60 2.037 25.465 8.301 530 O ARG 60 2.521 26.582 8.083531 N LEU 61 1.661 24.633 7.346 533 CA LEU 61 1.677 25.024 5.929 534 CBLEU 61 2.781 24.264 5.195 535 CG LEU 61 4.172 24.567 5.751 536 CD1 LEU61 5.239 23.709 5.080 537 CD2 LEU 61 4.515 26.046 5.613 538 C LEU 610.320 24.711 5.300 539 O LEU 61 −0.362 23.771 5.721 540 N ARG 62 −0.09925.514 4.340 542 CA ARG 62 −1.389 25.242 3.696 543 CB ARG 62 −2.38226.360 3.979 544 CG ARG 62 −3.774 25.940 3.515 545 CD ARG 62 −4.80427.039 3.737 546 NE ARG 62 −4.488 28.219 2.920 547 CZ ARG 62 −5.23328.603 1.881 548 NH1 ARG 62 −4.894 29.690 1.184 549 NH2 ARG 62 −6.32027.905 1.543 550 C ARG 62 −1.234 25.083 2.193 551 O ARG 62 −1.238 26.0601.435 552 N ILE 63 −1.078 23.840 1.781 554 CA ILE 63 −0.983 23.526 0.360555 CB ILE 63 0.271 22.682 0.139 556 CG2 ILE 63 0.477 22.375 −1.341 557CG1 ILE 63 1.497 23.395 0.698 558 CD1 ILE 63 2.764 22.580 0.472 559 CILE 63 −2.228 22.761 −0.077 560 O ILE 63 −2.542 21.699 0.468 561 N PRO64 −2.985 23.354 −0.984 562 CA PRO 64 −4.024 22.601 −1.685 563 CB PRO 64−4.708 23.605 −2.561 564 CG PRO 64 −3.951 24.926 −2.494 565 CD PRO 64−2.806 24.706 −1.518 566 C PRO 64 −3.379 21.493 −2.507 567 O PRO 64−2.364 21.730 −3.168 568 N ASP 65 −3.939 20.296 −2.440 570 CA ASP 65−3.409 19.158 −3.207 571 CB ASP 65 −3.961 17.880 −2.587 572 CG ASP 65−3.129 16.657 −2.960 573 OD1 ASP 65 −1.912 16.755 −2.874 574 OD2 ASP 65−3.722 15.600 −3.116 575 C ASP 65 −3.880 19.277 −4.653 576 O ASP 65−4.968 18.808 −5.005 577 N PHE 66 −3.050 19.893 −5.477 579 CA PHE 66−3.494 20.269 −6.821 580 CB PHE 66 −4.181 21.629 −6.695 581 CG PHE 66−5.613 21.721 −7.222 582 CD1 PHE 66 −6.416 20.590 −7.277 583 CE1 PHE 66−7.717 20.683 −7.752 584 CZ PHE 66 −8.217 21.909 −8.172 585 CE2 PHE 66−7.415 23.041 −8.118 586 CD2 PHE 66 −6.113 22.947 −7.643 587 C PHE 66−2.337 20.377 −7.812 588 O PHE 66 −1.347 19.640 −7.747 589 N CYS 67−2.561 21.237 −8.791 591 CA CYS 67 −1.571 21.560 −9.826 592 CB CYS 67−2.312 21.774 −11.140 593 SG CYS 67 −3.226 20.341 −11.759 594 C CYS 67−0.645 22.773 −9.551 595 O CYS 67 0.514 22.664 −9.967 596 N PRO 68−1.078 23.906 −8.989 597 CA PRO 68 −0.109 24.980 −8.721 598 CB PRO 68−0.885 26.113 −8.125 599 CG PRO 68 −2.351 25.727 −8.029 600 CD PRO 68−2.447 24.330 −8.616 601 C PRO 68 1.012 24.535 −7.778 602 O PRO 68 0.77123.980 −6.702 603 N PRO 69 2.231 24.787 −8.225 604 CA PRO 69 3.43924.392 −7.504 605 CB PRO 69 4.555 24.571 −8.489 606 CG PRO 69 4.02525.281 −9.725 607 CD PRO 69 2.533 25.445 −9.503 608 C PRO 69 3.70625.261 −6.280 609 O PRO 69 3.349 26.443 −6.249 610 N ALA 70 4.354 24.672−5.290 612 CA ALA 70 4.856 25.466 −4.159 613 CB ALA 70 3.961 25.221−2.950 614 C ALA 70 6.309 25.120 −3.811 615 O ALA 70 6.576 24.693 −2.679616 N PRO 71 7.255 25.506 −4.662 617 CA PRO 71 8.610 24.941 −4.572 618CB PRO 71 9.249 25.253 −5.890 619 CG PRO 71 8.358 26.208 −6.666 620 CDPRO 71 7.097 26.373 −5.838 621 C PRO 71 9.457 25.507 −3.429 622 O PRO 7110.352 24.812 −2.935 623 N ASP 72 9.002 26.597 −2.831 625 CA ASP 729.733 27.239 −1.734 626 CB ASP 72 9.277 28.691 −1.633 627 CG ASP 729.459 29.396 −2.975 628 OD1 ASP 72 10.596 29.691 −3.310 629 OD2 ASP 728.482 29.469 −3.709 630 C ASP 72 9.479 26.539 −0.399 631 O ASP 72 10.26626.686 0.545 632 N GLN 73 8.515 25.630 −0.392 634 CA GLN 73 8.223 24.8630.814 635 CB GLN 73 6.791 24.343 0.714 636 CG GLN 73 5.768 25.470 0.539637 CD GLN 73 5.306 26.071 1.870 638 OE1 GLN 73 4.161 25.855 2.285 639NE2 GLN 73 6.160 26.870 2.487 642 C GLN 73 9.197 23.698 0.972 643 O GLN73 9.472 23.314 2.114 644 N ILE 74 9.939 23.397 −0.087 646 CA ILE 7410.915 22.303 −0.062 647 CB ILE 74 11.453 22.127 −1.479 648 CG2 ILE 7412.705 21.258 −1.491 649 CG1 ILE 74 10.383 21.558 −2.402 650 CD1 ILE 749.911 20.188 −1.931 651 C ILE 74 12.084 22.586 0.874 652 O ILE 74 12.37421.751 1.739 653 N ASP 75 12.518 23.837 0.916 655 CA ASP 75 13.67624.194 1.739 656 CB ASP 75 14.085 25.627 1.420 657 CG ASP 75 14.50825.755 −0.040 658 OD1 ASP 75 15.667 25.482 −0.319 659 OD2 ASP 75 13.66226.090 −0.859 660 C ASP 75 13.331 24.091 3.216 661 O ASP 75 13.86323.201 3.895 662 N ARG 76 12.180 24.659 3.538 664 CA ARG 76 11.71724.740 4.920 665 CB ARG 76 10.475 25.618 4.901 666 CG ARG 76 9.93525.918 6.291 667 CD ARG 76 8.634 26.701 6.173 668 NE ARG 76 8.807 27.8215.234 669 CZ ARG 76 7.935 28.822 5.103 670 NH1 ARG 76 6.846 28.865 5.873671 NH2 ARG 76 8.161 29.790 4.212 672 C ARG 76 11.348 23.368 5.467 673 OARG 76 11.873 22.972 6.514 674 N PHE 77 10.748 22.554 4.614 676 CA PHE77 10.268 21.240 5.032 677 CB PHE 77 9.382 20.707 3.914 678 CG PHE 778.430 19.598 4.334 679 CD1 PHE 77 8.884 18.293 4.474 680 CE1 PHE 778.006 17.290 4.860 681 CZ PHE 77 6.673 17.595 5.102 682 CE2 PHE 77 6.21918.900 4.964 683 CD2 PHE 77 7.098 19.902 4.581 684 C PHE 77 11.42420.274 5.267 685 O PHE 77 11.458 19.614 6.316 686 N VAL 78 12.473 20.4084.473 688 CA VAL 78 13.632 19.536 4.635 689 CB VAL 78 14.473 19.6153.367 690 CG1 VAL 78 15.853 19.014 3.576 691 CG2 VAL 78 13.757 18.9392.202 692 C VAL 78 14.455 19.929 5.856 693 O VAL 78 14.808 19.037 6.638694 N GLN 79 14.423 21.203 6.209 696 CA GLN 79 15.146 21.665 7.396 697CB GLN 79 15.285 23.178 7.277 698 CG GLN 79 16.061 23.517 6.009 699 CDGLN 79 16.031 25.015 5.719 700 OE1 GLN 79 14.984 25.590 5.394 701 NE2GLN 79 17.208 25.613 5.755 704 C GLN 79 14.416 21.280 8.686 705 O GLN 7915.067 20.794 9.622 706 N ILE 80 13.097 21.183 8.602 708 CA ILE 8012.284 20.753 9.747 709 CB ILE 80 10.817 20.972 9.397 710 CG2 ILE 809.919 20.421 10.499 711 CG1 ILE 80 10.516 22.443 9.152 712 CD1 ILE 809.114 22.627 8.579 713 C ILE 80 12.488 19.270 10.043 714 O ILE 80 12.86218.908 11.167 715 N VAL 81 12.518 18.467 8.992 717 CA VAL 81 12.67017.025 9.186 718 CB VAL 81 12.057 16.315 7.982 719 CG1 VAL 81 11.95814.814 8.221 720 CG2 VAL 81 10.663 16.866 7.705 721 C VAL 81 14.13916.643 9.406 722 O VAL 81 14.412 15.635 10.075 723 N ASP 82 15.04817.551 9.078 725 CA ASP 82 16.461 17.376 9.429 726 CB ASP 82 17.32618.363 8.648 727 CG ASP 82 17.488 17.928 7.199 728 OD1 ASP 82 17.50416.727 6.966 729 OD2 ASP 82 17.708 18.792 6.360 730 C ASP 82 16.69717.631 10.910 731 O ASP 82 17.466 16.889 11.527 732 N GLU 83 15.87018.464 11.518 734 CA GLU 83 16.013 18.745 12.946 735 CB GLU 83 15.26920.046 13.228 736 CG GLU 83 15.349 20.464 14.690 737 CD GLU 83 14.55921.754 14.880 738 OE1 GLU 83 14.901 22.511 15.777 739 OE2 GLU 83 13.64621.976 14.098 740 C GLU 83 15.438 17.602 13.783 741 O GLU 83 16.04617.209 14.788 742 N ALA 84 14.451 16.916 13.228 744 CA ALA 84 13.87115.755 13.911 745 CB ALA 84 12.575 15.391 13.206 746 C ALA 84 14.80614.550 13.871 747 O ALA 84 15.153 14.012 14.931 748 N ASN 85 15.41114.347 12.707 750 CA ASN 85 16.353 13.237 12.467 751 CB ASN 85 16.37212.885 10.974 752 CG ASN 85 15.164 12.053 10.524 753 OD1 ASN 85 15.20410.815 10.530 754 ND2 ASN 85 14.154 12.735 10.018 757 C ASN 85 17.79113.547 12.906 758 O ASN 85 18.686 12.721 12.693 759 N ALA 86 18.02414.735 13.447 761 CA ALA 86 19.339 15.068 14.003 762 CB ALA 86 19.55916.574 13.918 763 C ALA 86 19.378 14.625 15.458 764 O ALA 86 20.44514.404 16.043 765 N ARG 87 18.191 14.498 16.024 767 CA ARG 87 18.02913.842 17.315 768 CB ARG 87 17.136 14.699 18.198 769 CG ARG 87 17.71116.094 18.400 770 CD ARG 87 16.802 16.924 19.298 771 NE ARG 87 16.59316.243 20.586 772 CZ ARG 87 17.076 16.694 21.746 773 NH1 ARG 87 16.87115.999 22.867 774 NH2 ARG 87 17.786 17.824 21.781 775 C ARG 87 17.37712.491 17.060 776 O ARG 87 17.181 12.099 15.905 777 N GLY 88 17.01011.796 18.120 779 CA GLY 88 16.302 10.521 17.947 780 C GLY 88 14.80510.710 18.177 781 O GLY 88 14.192 10.003 18.983 782 N GLU 89 14.22611.672 17.477 784 CA GLU 89 12.828 12.027 17.743 785 CB GLU 89 12.71613.531 17.973 786 CG GLU 89 13.759 14.080 18.946 787 CD GLU 89 13.79713.327 20.275 788 OE1 GLU 89 14.897 12.949 20.657 789 OE2 GLU 89 12.73913.072 20.833 790 C GLU 89 11.940 11.641 16.570 791 O GLU 89 12.28411.894 15.410 792 N ALA 90 10.773 11.102 16.880 794 CA ALA 90 9.84510.719 15.815 795 CB ALA 90 8.736 9.857 16.394 796 C ALA 90 9.269 11.94615.115 797 O ALA 90 8.989 12.982 15.737 798 N VAL 91 9.166 11.840 13.804800 CA VAL 91 8.658 12.956 13.004 801 CB VAL 91 9.792 13.517 12.152 802CG1 VAL 91 10.618 12.419 11.497 803 CG2 VAL 91 9.305 14.542 11.133 804 CVAL 91 7.457 12.546 12.158 805 O VAL 91 7.563 11.827 11.152 806 N GLY 926.315 13.060 12.572 808 CA GLY 92 5.055 12.760 11.904 809 C GLY 92 4.56013.976 11.135 810 O GLY 92 4.434 15.084 11.668 811 N VAL 93 4.406 13.7909.841 813 CA VAL 93 3.872 14.873 9.021 814 CB VAL 93 4.878 15.275 7.954815 CG1 VAL 93 4.323 16.435 7.141 816 CG2 VAL 93 6.217 15.661 8.574 817C VAL 93 2.570 14.426 8.381 818 O VAL 93 2.539 13.434 7.641 819 N HIS 941.511 15.143 8.703 821 CA HIS 94 0.194 14.792 8.202 822 CB HIS 94 −0.77914.660 9.372 823 CG HIS 94 −1.184 15.959 10.035 824 ND1 HIS 94 −2.31616.649 9.800 826 CE1 HIS 94 −2.336 17.748 10.579 827 NE2 HIS 94 −1.20517.747 11.320 828 CD2 HIS 94 −0.487 16.649 10.998 829 C HIS 94 −0.33815.827 7.226 830 O HIS 94 0.070 16.997 7.170 831 N CYS 95 −1.251 15.3416.417 833 CA CYS 95 −2.046 16.220 5.574 834 CB CYS 95 −1.582 16.1004.130 835 SG CYS 95 −1.201 14.431 3.563 836 C CYS 95 −3.508 15.855 5.781837 O CYS 95 −3.951 15.736 6.927 838 N ALA 96 −4.250 15.709 4.701 840 CAALA 96 −5.637 15.267 4.821 841 CB ALA 96 −6.441 15.866 3.673 842 C ALA96 −5.736 13.746 4.769 843 O ALA 96 −6.041 13.096 5.777 844 N LEU 97−5.350 13.195 3.629 846 CA LEU 97 −5.541 11.761 3.364 847 CB LEU 97−6.061 11.618 1.938 848 CG LEU 97 −7.424 12.274 1.757 849 CD1 LEU 97−7.826 12.289 0.287 850 CD2 LEU 97 −8.485 11.578 2.603 851 C LEU 97−4.289 10.893 3.491 852 O LEU 97 −4.369 9.692 3.221 853 N GLY 98 −3.16011.471 3.863 855 CA GLY 98 −1.905 10.712 3.845 856 C GLY 98 −1.42310.513 2.406 857 O GLY 98 −0.999 9.419 2.015 858 N PHE 99 −1.469 11.5941.641 860 CA PHE 99 −1.251 11.547 0.182 861 CB PHE 99 −2.616 11.474−0.511 862 CG PHE 99 −3.192 10.104 −0.885 863 CD1 PHE 99 −3.878 9.981−2.086 864 CE1 PHE 99 −4.417 8.757 −2.460 865 CZ PHE 99 −4.273 7.652−1.632 866 CE2 PHE 99 −3.591 7.773 −0.428 867 CD2 PHE 99 −3.052 8.998−0.056 868 C PHE 99 −0.557 12.796 −0.380 869 O PHE 99 −0.048 13.6640.348 870 N GLY 100 −0.417 12.749 −1.698 872 CA GLY 100 −0.103 13.897−2.577 873 C GLY 100 1.075 14.775 −2.168 874 O GLY 100 2.244 14.396−2.322 875 N ARG 101 0.729 15.928 −1.614 877 CA ARG 101 1.686 16.936−1.135 878 CB ARG 101 0.927 17.888 −0.223 879 CG ARG 101 −0.201 18.623−0.920 880 CD ARG 101 −1.283 18.948 0.098 881 NE ARG 101 −1.896 17.7000.576 882 CZ ARG 101 −3.186 17.589 0.898 883 NH1 ARG 101 −3.956 18.6770.959 884 NH2 ARG 101 −3.684 16.400 1.241 885 C ARG 101 2.789 16.356−0.269 886 O ARG 101 3.971 16.474 −0.612 887 N THR 102 2.407 15.5400.697 889 CA THR 102 3.390 15.105 1.682 890 CB THR 102 2.679 14.8252.994 891 OG1 THR 102 1.644 15.784 3.158 892 CG2 THR 102 3.654 14.9844.147 893 C THR 102 4.169 13.881 1.208 894 O THR 102 5.317 13.690 1.626895 N GLY 103 3.672 13.246 0.157 897 CA GLY 103 4.393 12.138 −0.478 898C GLY 103 5.542 12.723 −1.279 899 O GLY 103 6.702 12.320 −1.116 900 NTHR 104 5.230 13.846 −1.901 902 CA THR 104 6.198 14.624 −2.667 903 CBTHR 104 5.406 15.745 −3.332 904 OG1 THR 104 4.456 15.144 −4.200 905 CG2THR 104 6.276 16.698 −4.141 906 C THR 104 7.271 15.227 −1.762 907 O THR104 8.468 15.074 −2.041 908 N MET 105 6.860 15.660 −0.581 910 CA MET 1057.804 16.223 0.387 911 CB MET 105 7.004 16.865 1.511 912 CG MET 1056.267 18.112 1.045 913 SD MET 105 7.320 19.480 0.521 914 CE MET 1056.036 20.703 0.184 915 C MET 105 8.737 15.175 0.990 916 O MET 105 9.94915.417 1.065 917 N LEU 106 8.240 13.964 1.181 919 CA LEU 106 9.07612.898 1.738 920 CB LEU 106 8.159 11.785 2.234 921 CG LEU 106 8.94910.640 2.858 922 CD1 LEU 106 9.734 11.113 4.078 923 CD2 LEU 106 8.0179.497 3.231 924 C LEU 106 10.039 12.341 0.691 925 O LEU 106 11.22412.154 1.000 926 N ALA 107 9.622 12.368 −0.565 928 CA ALA 107 10.49111.911 −1.651 929 CB ALA 107 9.668 11.787 −2.928 930 C ALA 107 11.62912.893 −1.882 931 O ALA 107 12.796 12.478 −1.891 932 N CYS 108 11.32314.173 −1.741 934 CA CYS 108 12.341 15.206 −1.914 935 CB CYS 108 11.64216.542 −2.105 936 SG CYS 108 12.748 17.934 −2.406 937 C CYS 108 13.27715.298 −0.712 938 O CYS 108 14.475 15.541 −0.900 939 N TYR 109 12.80914.873 0.451 941 CA TYR 109 13.672 14.839 1.633 942 CB TYR 109 12.79814.666 2.871 943 CG TYR 109 13.600 14.590 4.165 944 CD1 TYR 109 14.16915.746 4.682 945 CE1 TYR 109 14.911 15.689 5.851 946 CZ TYR 109 15.08514.477 6.503 947 OH TYR 109 15.816 14.431 7.670 948 CE2 TYR 109 14.51513.318 5.992 949 CD2 TYR 109 13.772 13.376 4.821 950 C TYR 109 14.66713.685 1.554 951 O TYR 109 15.853 13.881 1.855 952 N LEU 110 14.25212.592 0.935 954 CA LEU 110 15.150 11.449 0.760 955 CB LEU 110 14.32510.233 0.355 956 CG LEU 110 13.330 9.830 1.438 957 CD1 LEU 110 12.3948.732 0.941 958 CD2 LEU 110 14.044 9.397 2.714 959 C LEU 110 16.19111.738 −0.315 960 O LEU 110 17.384 11.472 −0.104 961 N VAL 111 15.79912.500 −1.320 963 CA VAL 111 16.768 12.884 −2.343 964 CB VAL 111 16.05013.510 −3.534 965 CG1 VAL 111 17.044 13.820 −4.649 966 CG2 VAL 11114.951 12.599 −4.065 967 C VAL 111 17.788 13.872 −1.788 968 O VAL 11118.968 13.511 −1.681 969 N LYS 112 17.289 14.906 −1.131 971 CA LYS 11218.133 16.028 −0.706 972 CB LYS 112 17.180 17.183 −0.414 973 CG LYS 11217.884 18.500 −0.111 974 CD LYS 112 16.855 19.613 0.058 975 CE LYS 11217.500 20.951 0.399 976 NZ LYS 112 16.473 21.985 0.600 977 C LYS 11218.991 15.749 0.529 978 O LYS 112 20.076 16.325 0.655 979 N GLU 11318.579 14.836 1.393 981 CA GLU 113 19.397 14.591 2.583 982 CB GLU 11318.529 14.748 3.822 983 CG GLU 113 17.987 16.166 3.915 984 CD GLU 11319.126 17.184 3.998 985 OE1 GLU 113 20.005 16.998 4.829 986 OE2 GLU 11319.035 18.179 3.292 987 C GLU 113 20.068 13.226 2.605 988 O GLU 11321.094 13.067 3.275 989 N ARG 114 19.526 12.261 1.882 991 CA ARG 11420.133 10.929 1.913 992 CB ARG 114 19.027 9.877 1.987 993 CG ARG 11418.007 10.100 3.109 994 CD ARG 114 18.468 9.660 4.504 995 NE ARG 11419.401 10.604 5.145 996 CZ ARG 114 19.035 11.496 6.069 997 NH1 ARG 11419.929 12.362 6.549 998 NH2 ARG 114 17.764 11.557 6.473 999 C ARG 11420.981 10.678 0.673 1000 O ARG 114 21.785 9.739 0.645 1001 N GLY 11520.796 11.510 −0.341 1003 CA GLY 115 21.510 11.329 −1.609 1004 C GLY 11520.837 10.222 −2.414 1005 O GLY 115 21.470 9.522 −3.214 1006 N LEU 11619.546 10.079 −2.178 1008 CA LEU 116 18.772 8.997 −2.773 1009 CB LEU 11617.727 8.589 −1.744 1010 CG LEU 116 16.894 7.413 −2.217 1011 CD1 LEU 11617.791 6.206 −2.449 1012 CD2 LEU 116 15.804 7.092 −1.207 1013 C LEU 11618.073 9.479 −4.032 1014 O LEU 116 17.284 10.424 −3.967 1015 N ALA 11718.279 8.782 −5.138 1017 CA ALA 117 17.611 9.152 −6.394 1018 CB ALA 11717.987 8.151 −7.478 1019 C ALA 117 16.095 9.176 −6.220 1020 O ALA 11715.533 8.384 −5.450 1021 N ALA 118 15.436 9.995 −7.025 1023 CA ALA 11813.988 10.211 −6.878 1024 CB ALA 118 13.582 11.375 −7.772 1025 C ALA 11813.158 8.978 −7.230 1026 O ALA 118 12.188 8.680 −6.523 1027 N GLY 11913.708 8.131 −8.087 1029 CA GLY 119 13.088 6.840 −8.399 1030 C GLY 11913.061 5.926 −7.175 1031 O GLY 119 11.993 5.425 −6.807 1032 N ASP 12014.148 5.926 −6.418 1034 CA ASP 120 14.251 5.042 −5.254 1035 CB ASP 12015.707 4.948 −4.815 1036 CG ASP 120 16.663 4.621 −5.955 1037 OD1 ASP 12017.707 5.261 −6.008 1038 OD2 ASP 120 16.324 3.788 −6.785 1039 C ASP 12013.448 5.601 −4.081 1040 O ASP 120 12.790 4.829 −3.370 1041 N ALA 12113.314 6.918 −4.028 1043 CA ALA 121 12.517 7.550 −2.975 1044 CB ALA 12112.743 9.056 −3.034 1045 C ALA 121 11.037 7.244 −3.166 1046 O ALA 12110.409 6.689 −2.253 1047 N ILE 122 10.594 7.289 −4.413 1049 CA ILE 1229.197 6.973 −4.717 1050 CB ILE 122 8.869 7.495 −6.109 1051 CG2 ILE 1227.440 7.144 −6.508 1052 CG1 ILE 122 9.065 8.999 −6.192 1053 CD1 ILE 1228.752 9.479 −7.602 1054 C ILE 122 8.919 5.472 −4.668 1055 O ILE 1227.833 5.088 −4.224 1056 N ALA 123 9.935 4.647 −4.860 1058 CA ALA 1239.738 3.198 −4.754 1059 CB ALA 123 10.936 2.490 −5.375 1060 C ALA 1239.582 2.754 −3.303 1061 O ALA 123 8.662 1.985 −3.000 1062 N GLU 12410.257 3.447 −2.402 1064 CA GLU 124 10.142 3.136 −0.975 1065 CB GLU 12411.335 3.773 −0.276 1066 CG GLU 124 12.629 3.171 −0.810 1067 CD GLU 12413.834 3.993 −0.375 1068 OE1 GLU 124 13.702 4.737 0.586 1069 OE2 GLU 12414.844 3.928 −1.066 1070 C GLU 124 8.834 3.682 −0.410 1071 O GLU 1248.128 2.970 0.317 1072 N ILE 125 8.393 4.785 −0.991 1074 CA ILE 1257.097 5.367 −0.647 1075 CB ILE 125 7.051 6.755 −1.282 1076 CG2 ILE 1255.634 7.296 −1.424 1077 CG1 ILE 125 7.924 7.721 −0.492 1078 CD1 ILE 1257.832 9.129 −1.062 1079 C ILE 125 5.942 4.500 −1.142 1080 O ILE 1255.103 4.112 −0.321 1081 N ARG 126 6.119 3.899 −2.307 1083 CA ARG 1265.076 3.066 −2.908 1084 CB ARG 126 5.407 2.931 −4.391 1085 CG ARG 1264.329 2.180 −5.161 1086 CD ARG 126 2.960 2.828 −4.972 1087 NE ARG 1261.928 2.146 −5.771 1088 CZ ARG 126 1.204 1.105 −5.349 1089 NH1 ARG 1261.392 0.602 −4.126 1090 NH2 ARG 126 0.283 0.568 −6.151 1091 C ARG 1264.976 1.688 −2.251 1092 O ARG 126 3.871 1.139 −2.172 1093 N ARG 1276.031 1.259 −1.574 1095 CA ARG 127 5.951 0.002 −0.827 1096 CB ARG 1277.340 −0.599 −0.665 1097 CG ARG 127 7.992 −0.873 −2.013 1098 CD ARG 1279.253 −1.709 −1.840 1099 NE ARG 127 10.083 −1.187 −0.744 1100 CZ ARG 12711.278 −0.622 −0.922 1101 NH1 ARG 127 11.739 −0.405 −2.156 1102 NH2 ARG127 11.981 −0.212 0.135 1103 C ARG 127 5.323 0.199 0.551 1104 O ARG 1274.970 −0.785 1.212 1105 N LEU 128 5.150 1.443 0.966 1107 CA LEU 1284.425 1.710 2.207 1108 CB LEU 128 5.111 2.827 2.998 1109 CG LEU 1286.092 2.324 4.058 1110 CD1 LEU 128 5.422 1.304 4.972 1111 CD2 LEU 1287.379 1.752 3.473 1112 C LEU 128 2.978 2.110 1.920 1113 O LEU 128 2.0771.727 2.679 1114 N ARG 129 2.761 2.777 0.793 1116 CA ARG 129 1.427 3.2930.438 1117 CB AEG 129 1.031 4.282 1.537 1118 CG ARG 129 −0.411 4.7731.494 1119 CD ARG 129 −0.591 5.850 2.558 1120 NE ARG 129 −1.998 6.2222.754 1121 CZ ARG 129 −2.534 6.300 3.974 1122 NH1 ARG 129 −1.803 5.9705.040 1123 NH2 ARG 129 −3.810 6.660 4.124 1124 C ARG 129 1.450 4.008−0.923 1125 O ARG 129 2.368 4.778 −1.228 1126 N PRO 130 0.469 3.706−1.756 1127 CA PRO 130 0.213 4.511 −2.959 1128 CB PRO 130 −0.828 3.738−3.711 1129 CG PRO 130 −1.370 2.628 −2.821 1130 CD PRO 130 −0.549 2.674−1.544 1131 C PRO 130 −0.326 5.908 −2.632 1132 O PRO 130 −0.940 6.120−1.580 1133 N GLY 131 −0.088 6.856 −3.526 1135 CA GLY 131 −0.756 8.161−3.404 1136 C GLY 131 0.130 9.399 −3.532 1137 O GLY 131 0.387 10.084−2.535 1138 N SER 132 0.443 9.765 −4.765 1140 CA SER 132 1.174 11.013−5.045 1141 CB SER 132 2.674 10.743 −5.102 1142 OG SER 132 3.106 10.386−3.795 1143 C SER 132 0.702 11.627 −6.364 1144 O SER 132 0.463 10.910−7.342 1145 N ILE 133 0.544 12.940 −6.372 1147 CA ILE 133 0.050 13.633−7.571 1148 CB ILE 133 −0.575 14.966 −7.146 1149 CG2 ILE 133 −0.94515.872 −8.318 1150 CG1 ILE 133 −1.815 14.721 −6.301 1151 CD1 ILE 133−2.665 15.984 −6.247 1152 C ILE 133 1.173 13.830 −8.589 1153 O ILE 1332.241 14.357 −8.258 1154 N GLU 134 0.858 13.565 −9.851 1156 CA GLU 1341.848 13.637 −10.935 1157 CB GLU 134 1.226 13.055 −12.197 1158 CG GLU134 0.892 11.578 −12.033 1159 CD GLU 134 0.188 11.072 −13.288 1160 OE1GLU 134 0.284 11.753 −14.299 1161 OE2 GLU 134 −0.574 10.124 −13.159 1162C GLU 134 2.338 15.052 −11.246 1163 O GLU 134 3.504 15.195 −11.627 1164N THR 135 1.612 16.071 −10.814 1166 CA THR 135 2.082 17.439 −11.029 1167CB THR 135 0.925 18.406 −10.834 1168 OG1 THR 135 −0.146 18.009 −11.6781169 CG2 THR 135 1.340 19.819 −11.219 1170 C THR 135 3.200 17.777−10.045 1171 O THR 135 4.247 18.269 −10.482 1172 N TYR 136 3.145 17.168−8.871 1174 CA TYR 136 4.200 17.364 −7.872 1175 CB TYR 136 3.647 17.007−6.503 1176 CG TYR 136 2.593 17.957 −5.958 1177 CD1 TYR 136 2.754 19.331−6.085 1178 CE1 TYR 136 1.784 20.192 −5.589 1179 CZ TYR 136 0.662 19.673−4.956 1180 OH TYR 136 −0.365 20.508 −4.586 1181 CE2 TYR 136 0.51418.302 −4.802 1182 CD2 TYR 136 1.484 17.443 −5.299 1183 C TYR 136 5.40616.470 −8.149 1184 O TYR 136 6.541 16.839 −7.813 1185 N GLU 137 5.18015.442 −8.952 1187 CA GLU 137 6.265 14.549 −9.349 1188 CB GLU 137 5.71913.155 −9.656 1189 CG GLU 137 4.785 12.608 −8.578 1190 CD GLU 137 5.42212.575 −7.186 1191 OE1 GLU 137 6.135 11.623 −6.914 1192 OE2 GLU 1374.977 13.366 −6.368 1193 C GLU 137 6.973 15.096 −10.588 1194 O GLU 1378.138 14.762 −10.831 1195 N GLN 138 6.334 16.009 −11.297 1197 CA GLN 1387.038 16.697 −12.375 1198 CB GLN 138 6.020 17.242 −13.378 1199 CG GLN138 6.593 17.418 −14.789 1200 CD GLN 138 7.711 18.459 −14.852 1201 OE1GLN 138 7.527 19.615 −14.453 1202 NE2 GLN 138 8.856 18.036 −15.359 1205C GLN 138 7.830 17.832 −11.744 1206 O GLN 138 9.028 17.975 −12.020 1207N GLU 139 7.235 18.401 −10.710 1209 CA GLU 139 7.822 19.523 −9.979 1210CB GLU 139 6.784 20.076 −9.008 1211 CG GLU 139 5.631 20.789 −9.694 1212CD GLU 139 4.577 21.116 −8.643 1213 OE1 GLU 139 3.406 20.882 −8.904 1214OE2 GLU 139 4.978 21.486 −7.547 1215 C GLU 139 9.066 19.204 −9.155 1216O GLU 139 9.860 18.291 −9.445 1217 N LYS 140 9.013 19.819 −7.986 1219 CALYS 140 10.132 20.030 −7.062 1220 CB LYS 140 9.556 21.013 −6.045 1221 CGLYS 140 8.214 20.466 −5.559 1222 CD LYS 140 7.437 21.413 −4.657 1223 CELYS 140 6.136 20.760 −4.204 1224 NZ LYS 140 5.352 21.653 −3.340 1225 CLYS 140 10.659 18.811 −6.308 1226 O LYS 140 11.628 18.950 −5.556 1227 NALA 141 10.067 17.644 −6.490 1229 CA ALA 141 10.575 16.481 −5.770 1230CB ALA 141 9.434 15.828 −5.018 1231 C ALA 141 11.189 15.445 −6.686 1232O ALA 141 12.180 14.800 −6.321 1233 N VAL 142 10.646 15.313 −7.882 1235CA VAL 142 11.112 14.219 −8.731 1236 CB VAL 142 9.960 13.276 −9.049 1237CG1 VAL 142 10.451 12.006 −9.744 1238 CG2 VAL 142 9.205 12.915 −7.7771239 C VAL 142 11.793 14.739 −9.985 1240 O VAL 142 13.010 14.930 −9.9411241 N PHE 143 11.039 15.138 −10.999 1243 CA PHE 143 11.676 15.455−12.282 1244 CB PHE 143 10.634 15.597 −13.382 1245 CG PHE 143 9.95414.292 −13.786 1246 CD1 PHE 143 8.636 14.302 −14.227 1247 CE1 PHE 1438.014 13.113 −14.588 1248 CZ PHE 143 8.712 11.913 −14.515 1249 CE2 PHE143 10.034 11.906 −14.087 1250 CD2 PHE 143 10.656 13.095 −13.727 1251 CPHE 143 12.546 16.705 −12.210 1252 O PHE 143 13.767 16.565 −12.358 1253N GLN 144 12.013 17.787 −11.663 1255 CA GLN 144 12.795 19.027 −11.5821256 CB GLN 144 11.858 20.182 −11.266 1257 CG GLN 144 10.866 20.430−12.392 1258 CD GLN 144 9.865 21.499 −11.970 1259 OE1 GLN 144 9.97722.066 −10.877 1260 NE2 GLN 144 8.789 21.612 −12.730 1263 C GLE 14413.899 18.976 −10.530 1264 O GLN 144 14.992 19.499 −10.780 1265 N PHE145 13.747 18.123 −9.530 1267 CA PHE 145 14.789 18.046 −8.507 1268 CBPHE 145 14.174 17.614 −7.185 1269 CG PHE 145 15.117 17.799 −6.002 1270CD1 PHE 145 16.060 18.817 −6.028 1271 CE1 PHE 145 16.928 18.993 −4.9591272 CZ PHE 145 16.852 18.147 −3.862 1273 CE2 PHE 145 15.912 17.125−3.839 1274 CD2 PHE 145 15.046 16.948 −4.910 1275 C PHE 145 15.89617.074 −8.921 1276 O PHE 145 17.066 17.324 −8.618 1277 N TYR 146 15.57516.155 −9.813 1279 CA TYR 146 16.572 15.226 −10.339 1280 CB TYR 14615.851 13.970 −10.831 1281 CG TYR 146 16.737 12.742 −10.981 1282 CD1 TYR146 17.740 12.497 −10.051 1283 CE1 TYR 146 18.546 11.372 −10.179 1284 CZTYR 146 18.343 10.493 −11.234 1285 OH TYR 146 19.139 9.376 −11.360 1286CE2 TYR 146 17.339 10.735 −12.163 1287 CD2 TYR 146 16.533 11.860 −12.0341288 C TYR 146 17.338 15.873 −11.489 1289 O TYR 146 18.529 15.599−11.669 1290 N GLN 147 16.746 16.906 −12.070 1292 CA GLN 147 17.45317.732 −13.054 1293 CB GLN 147 16.406 18.449 −13.901 1294 CG GLN 14715.549 17.427 −14.643 1295 CD GLN 147 14.345 18.080 −15.314 1296 OE1 GLN147 13.487 18.690 −14.663 1297 NE2 GLN 147 14.234 17.837 −16.608 1300 CGLN 147 18.362 18.738 −12.344 1301 O GLN 147 19.451 19.052 −12.841 1302N ARG 148 18.048 18.993 −11.083 1304 CA ARG 148 18.876 19.832 −10.2131305 CB ARG 148 17.949 20.396 −9.142 1306 CG ARG 148 18.542 21.580−8.392 1307 CD ARG 148 17.557 22.055 −7.335 1308 NE ARG 148 16.19822.087 −7.900 1309 CZ ARG 148 15.087 22.121 −7.160 1310 NH1 ARG 14815.170 22.258 −5.835 1311 NH2 ARG 148 13.890 22.094 −7.754 1312 C ARG148 19.999 19.011 −9.562 1313 O ARG 148 21.002 19.576 −9.109 1314 N THR149 19.942 17.699 −9.743 1316 CA THR 149 20.972 16.781 −9.236 1317 CBTHR 149 20.338 15.401 −9.049 1318 OG1 THR 149 19.179 15.553 −8.246 1319CG2 THR 149 21.258 14.404 −8.349 1320 C THR 149 22.159 16.691 −10.2041321 O THR 149 23.207 16.135 −9.856 1322 N LYS 150 22.064 17.412 −11.3131324 CA LYS 150 23.145 17.484 −12.303 1325 CB LYS 150 22.552 18.209−13.509 1326 CG LYS 150 23.565 18.460 −14.618 1327 CD LYS 150 23.04219.495 −15.608 1328 CE LYS 150 22.778 20.830 −14.914 1329 NZ LYS 15024.010 21.383 −14.325 1330 C LYS 150 24.372 18.264 −11.805 1331 O LYS150 25.484 18.044 −12.301 1332 N GLU 151 24.202 19.068 −10.767 1334 CAGLU 151 25.341 19.791 −10.190 1335 CB GLU 151 24.824 21.021 −9.441 1336CG GLU 151 23.695 20.691 −8.469 1337 CD GLU 151 23.111 21.967 −7.8721338 OE1 GLU 151 23.517 22.322 −6.775 1339 OE2 GLU 151 22.311 22.593−8.553 1340 C GLU 151 26.191 18.878 −9.295 1341 O GLU 151 26.041 18.951−8.083 1342 OXT GLU 151 27.093 18.256 −9.838

TABLE X Atom Atom Residue No name Residue No x coord y coord z coord1271 N PRO 159 7.810 59.922 28.682 1272 CA PRO 159 7.834 60.673 27.4241273 CB PRO 159 6.519 61.385 27.362 1274 CG PRO 159 5.766 61.156 28.6641275 CD PRO 159 6.652 60.259 29.513 1276 C PRO 159 9.003 61.649 27.4191277 O PRO 159 9.148 62.480 28.324 1278 N THR 160 9.817 61.560 26.3861280 CA THR 160 11.063 62.328 26.377 1281 CB THR 160 12.168 61.44025.831 1282 OG1 THR 160 12.161 60.233 26.582 1283 CG2 THR 160 13.52562.113 25.990 1284 C THR 160 10.970 63.589 25.534 1285 O THR 160 10.73863.526 24.323 1286 N ARG 161 11.150 64.726 26.181 1288 CA ARG 161 11.18366.001 25.462 1289 CB ARG 161 11.339 67.120 26.484 1290 CG ARG 16111.208 68.500 25.851 1291 CD ARG 161 11.629 69.598 26.819 1292 NE ARG161 13.058 69.463 27.148 1293 CZ ARG 161 13.521 69.226 28.379 1294 NH1ARG 161 12.672 69.116 29.403 1295 NH2 ARG 161 14.834 69.100 28.583 1296C ARG 161 12.376 66.009 24.511 1297 O ARG 161 13.487 65.624 24.893 1298N ILE 162 12.111 66.309 23.252 1300 CA ILE 162 13.177 66.368 22.253 1301CB ILE 162 12.753 65.567 21.024 1302 CG2 ILE 162 13.842 65.603 19.9581303 CG1 ILE 162 12.418 64.122 21.382 1304 CD1 ILE 162 13.635 63.35621.892 1305 C ILE 162 13.402 67.823 21.869 1306 O ILE 162 14.536 68.28121.682 1307 N LEU 163 12.300 68.549 21.819 1309 CA LEU 163 12.333 69.98821.536 1310 CB LEU 163 11.808 70.243 20.122 1311 CG LEU 163 12.83569.894 19.050 1312 CD1 LEU 163 12.244 70.054 17.656 1313 CD2 LEU 16314.079 70.762 19.197 1314 C LEU 163 11.466 70.721 22.550 1315 O LEU 16310.632 70.092 23.213 1316 N PRO 164 11.703 72.012 22.725 1317 CA PRO 16410.737 72.843 23.446 1318 CB PRO 164 11.263 74.242 23.358 1319 CG PRO164 12.565 74.238 22.571 1320 CD PRO 164 12.804 72.796 22.156 1321 C PRO164 9.354 72.708 22.817 1322 O PRO 164 9.170 72.931 21.615 1323 N ASN165 8.421 72.281 23.654 1325 CA ASN 165 7.035 71.973 23.265 1326 CB ASN165 6.370 73.188 22.628 1327 CG ASN 165 6.228 74.328 23.628 1328 OD1 ASN165 5.554 74.179 24.654 1329 ND2 ASN 165 6.774 75.473 23.259 1332 C ASN165 6.920 70.781 22.313 1333 O ASN 165 6.005 70.751 21.479 1334 N LEU166 7.743 69.766 22.528 1336 CA LEU 166 7.694 68.542 21.715 1337 CB LEU166 8.507 68.743 20.438 1338 CG LEU 166 8.613 67.459 19.613 1339 CD1 LEU166 7.243 66.934 19.198 1340 CD2 LEU 166 9.495 67.656 18.386 1341 C LEU166 8.242 67.343 22.489 1342 O LEU 166 9.458 67.203 22.685 1343 N TYR167 7.326 66.479 22.889 1345 CA TYR 167 7.661 65.244 23.606 1346 CB TYR167 6.751 65.099 24.825 1347 CG TYR 167 7.047 66.005 26.019 1348 CD1 TYR167 6.568 67.309 26.051 1349 CB1 TYR 167 6.835 68.120 27.149 1350 CZ TYR167 7.573 67.620 28.213 1351 OH TYR 167 7.776 68.406 29.326 1352 CB2 TYR167 8.046 66.316 28.186 1353 CD2 TYR 167 7.779 65.508 27.090 1354 C TYR167 7.477 64.016 22.714 1355 O TYR 167 6.441 63.841 22.060 1356 N LEU168 8.487 63.166 22.723 1358 CA LEU 168 8.456 61.889 22.009 1359 CB LEU168 9.904 61.458 21.802 1360 CG LEU 168 10.031 60.174 20.994 1361 CD1LEU 168 9.446 60.354 19.600 1362 CD2 LEU 168 11.490 59.745 20.909 1363 CLEU 168 7.727 60.843 22.848 1364 O LEU 168 8.101 60.587 24.002 1365 NGLY 169 6.710 60.237 22.261 1367 CA GLY 169 5.895 59.257 22.975 1368 CGLY 169 5.992 57.843 22.405 1369 O GLY 169 5.272 57.451 21.476 1370 NCYS 170 6.877 57.075 23.011 1372 CA CYS 170 6.962 55.637 22.743 1373 CBCYS 170 8.266 55.120 23.348 1374 SG CYS 170 8.700 53.387 23.049 1375 CCYS 170 5.772 54.968 23.422 1376 O CYS 170 5.354 55.430 24.490 1377 NGLN 171 5.388 53.797 22.933 1379 CA GLN 171 4.230 53.025 23.430 1380 CBGLN 171 4.108 51.825 22.499 1381 CG GLN 171 5.387 50.998 22.578 1382 CDGLN 171 5.407 49.864 21.564 1383 OE1 GLN 171 6.432 49.650 20.907 1384NE2 GLN 171 4.271 49.211 21.394 1387 C GLN 171 4.332 52.480 24.867 1388O GLN 171 3.398 51.816 25.329 1389 N ARG 172 5.436 52.731 25.555 1391 CAARG 172 5.604 52.289 26.939 1392 CB ARG 172 7.038 51.800 27.117 1393 CGARG 172 7.400 50.730 26.092 1394 CD ARG 172 6.526 49.488 26.229 1395 NEARG 172 6.825 48.529 25.157 1396 CZ ARG 172 6.807 47.207 25.336 1397 NH1ARG 172 6.506 46.701 26.535 1398 NH2 ARG 172 7.090 46.392 24.318 1399 CARG 172 5.342 53.446 27.903 1400 O ARG 172 5.244 53.239 29.119 1401 NASP 173 5.183 54.637 27.348 1403 CA ASP 173 4.962 55.838 28.160 1404 CBASP 173 5.188 57.086 27.313 1405 CG ASP 173 6.621 57.152 26.808 1406 OD1ASP 173 7.488 56.556 27.438 1407 OD2 ASP 173 6.857 57.886 25.856 1408 CASP 173 3.558 55.868 28.742 1409 O ASP 173 2.586 55.445 28.105 1410 NVAL 174 3.467 56.365 29.962 1412 CA VAL 174 2.163 56.446 30.630 1413 CBVAL 174 2.373 56.566 32.139 1414 CG1 VAL 174 1.055 56.735 32.891 1415CG2 VAL 174 3.125 55.356 32.675 1416 C VAL 174 1.364 57.639 30.113 1417O VAL 174 1.762 58.797 30.283 1418 N LEU 175 0.206 57.346 29.543 1420 CALEU 175 −0.702 58.394 29.049 1421 CB LEU 175 −1.527 57.836 27.897 1422CG LEU 175 −0.675 57.638 26.651 1423 CD1 LEU 175 −1.479 56.974 25.5401424 CD2 LEU 175 −0.108 58.972 26.178 1425 C LEU 175 −1.645 58.91130.134 1426 O LEU 175 −2.867 58.748 30.049 1427 N ASN 176 −1.067 59.50531.162 1429 CA ASN 176 −1.863 60.067 32.249 1430 CB ASN 176 −1.08159.893 33.548 1431 CG ASN 176 −1.887 60.398 34.738 1432 OD1 ASN 176−1.914 61.603 35.013 1433 ND2 ASN 176 −2.584 59.485 35.390 1436 C ASN176 −2.126 61.537 31.950 1437 O ASN 176 −1.178 62.322 31.834 1438 N LYS177 −3.386 61.939 31.994 1440 CA LYS 177 −3.742 63.305 31.584 1441 CBLYS 177 −5.253 63.389 31.401 1442 CG LYS 177 −5.678 64.803 31.020 1443CD LYS 177 −7.140 64.870 30.596 1444 CE LYS 177 −7.372 64.142 29.2771445 NZ LYS 177 −8.756 64.328 28.811 1446 C LYS 177 −3.261 64.383 32.5561447 O LYS 177 −2.667 65.360 32.080 1448 N GLU 178 −3.132 64.034 33.8271450 CA GLU 178 −2.633 64.996 34.812 1451 CB GLU 178 −2.907 64.46936.214 1452 CG GLU 178 −2.362 65.422 37.273 1453 CD GLU 178 −2.32664.727 38.627 1454 OE1 GLU 178 −2.944 63.675 38.737 1455 OE2 GLU 178−1.735 65.281 39.546 1456 C GLU 178 −1.130 65.173 34.651 1457 O GLU 178−0.668 66.316 34.563 1458 N LEU 179 −0.466 64.094 34.269 1460 CA LEU 1790.980 64.110 34.041 1461 CB LEU 179 1.442 62.650 34.005 1462 CG LEU 1792.927 62.451 33.702 1463 CD1 LEU 179 3.498 61.304 34.528 1464 CD2 LEU179 3.183 62.216 32.213 1465 C LEU 179 1.329 64.845 32.748 1466 O LEU179 2.333 65.571 32.712 1467 N MET 180 0.413 64.856 31.796 1469 CA MET180 0.649 65.614 30.571 1470 CB MET 180 −0.261 65.095 29.465 1471 CG MET180 0.038 63.631 29.162 1472 SD MET 180 −0.821 62.933 27.733 1473 CE MET180 −2.525 63.329 28.179 1474 C MET 180 0.410 67.099 30.810 1475 O MET180 1.274 67.904 30.439 1476 N GLN 181 −0.508 67.410 31.710 1478 CA GLN181 −0.753 68.808 32.077 1479 CB GLN 181 −2.083 68.880 32.818 1480 CGGLN 181 −3.225 68.380 31.940 1481 CD GLN 181 −04.522 68.322 32.740 1482OE1 GLN 181 −4.974 67.247 33.159 1483 NE2 GLN 181 −5.108 69.488 32.9391486 C GLN 181 0.362 69.363 32.966 1487 O GLN 181 0.765 70.515 32.7741488 N GLN 182 1.029 68.482 33.697 1490 CA GLN 182 2.188 68.872 34.5131491 CB GLN 182 2.373 67.811 35.590 1492 CG GLN 182 1.165 67.769 36.5171493 CD GLN 182 1.232 66.556 37.438 1494 OE1 GLN 182 0.810 65.449 37.0751495 NE2 GLN 182 1.696 66.796 38.650 1498 C GLN 182 3.479 68.994 33.7001499 O GLN 182 4.458 69.573 34.183 1500 N ASN 183 3.465 68.490 32.4771502 CA ASN 183 4.597 68.659 31.560 1503 CB ASN 183 4.841 67.355 30.8041504 CG ASN 183 5.774 66.424 31.582 1505 OD1 ASN 183 6.998 66.487 31.4201506 ND2 ASN 183 5.195 65.551 32.387 1509 C ASN 183 4.345 69.797 30.5711510 O ASN 183 5.234 70.153 29.787 1511 N GLY 184 3.136 70.340 30.6001513 CA GLY 184 2.778 71.482 29.753 1514 C GLY 184 2.211 71.046 28.4061515 O GLY 184 2.220 71.826 27.445 1516 N ILE 185 1.753 69.807 28.3401518 CA ILE 185 1.241 69.238 27.090 1519 CB ILE 185 1.373 67.717 27.1641520 CG2 ILE 185 0.772 67.030 25.942 1521 CG1 ILE 185 2.837 67.32227.322 1522 CD1 ILE 185 3.009 65.809 27.389 1523 C ILE 185 −0.206 69.65526.863 1524 O ILE 185 −1.093 69.385 27.685 1525 N GLY 186 −0.429 70.29125.727 1527 CA GLY 186 −1.752 70.800 25.376 1528 C GLY 186 −2.207 70.18724.062 1529 O GLY 186 −3.407 70.157 23.753 1530 N TYR 187 −1.239 69.76723.267 1532 CA TYR 187 −1.544 69.081 22.011 1533 CB TYR 187 −0.90069.854 20.863 1534 CG TYR 187 −1.538 71.217 20.579 1535 CD1 TYR 187−1.014 72.377 21.140 1536 CE1 TYR 187 −1.600 73.607 20.873 1537 CZ TYR187 −2.710 73.675 20.042 1538 OH TYR 187 −3.299 74.893 19.786 1539 CE2TYR 187 −3.233 72.522 19.476 1540 CD2 TYR 187 −2.645 71.294 19.745 1541C TYR 187 −1.053 67.634 22.050 1542 O TYR 187 0.029 67.340 22.571 1543 NVAL 188 −1.887 66.726 21.576 1545 CA VAL 188 −1.518 65.303 21.553 1546CB VAL 188 −2.387 64.524 22.540 1547 CG1 VAL 188 −2.002 63.048 22.5531548 CG2 VAL 188 −2.292 65.097 23.950 1549 C VAL 188 −1.680 64.71720.151 1550 O VAL 188 −2.798 64.541 19.650 1551 N LEU 189 −0.558 64.35119.563 1553 CA LEU 189 −0.536 63.798 18.210 1554 CB LEU 189 0.669 64.41717.504 1555 CG LEU 189 0.605 64.373 15.979 1556 CD1 LEU 189 1.550 65.40815.384 1557 CD2 LEU 189 0.899 62.991 15.407 1558 C LEU 189 −0.410 62.27818.301 1559 O LEU 189 0.602 61.751 18.775 1560 N ASN 190 −1.431 61.58117.841 1562 CA ASN 190 −1.420 60.121 17.902 1563 CB ASN 190 −2.76059.630 18.441 1564 CG ASN 190 −2.789 58.109 18.367 1565 OD1 ASN 190−3.450 57.533 17.495 1566 ND2 ASN 190 −1.926 57.480 19.143 1569 C ASN190 −1.139 59.514 16.529 1570 O ASN 190 −1.955 59.608 15.606 1571 N ALA191 −0.025 58.806 16.447 1573 CA ALA 191 0.407 58.172 15.194 1574 CB ALA191 1.908 58.389 15.046 1575 C ALA 191 0.112 56.670 15.130 1576 O ALA191 0.899 55.919 14.542 1577 N SER 192 −0.958 56.224 15.766 1579 CA SER192 −1.206 54.782 15.854 1580 CB SER 192 −1.912 54.479 17.170 1581 OGSER 192 −1.059 54.926 18.225 1582 C SER 192 −1.992 54.260 14.649 1583 OSER 192 −2.502 55.028 13.822 1584 N ASN 193 −1.985 52.944 14.516 1586 CAASN 193 −2.607 52.290 13.363 1587 CB ASN 193 −2.303 50.793 13.362 1588CG ASN 193 −2.461 50.148 14.738 1589 OD1 ASN 193 −3.403 50.423 15.4901590 ND2 ASN 193 −1.525 49.268 15.045 1593 C ASN 193 −4.102 52.54613.265 1594 O ASN 193 −4.865 52.431 14.231 1595 N THR 194 −4.471 52.92012.050 1597 CA THR 194 −5.833 53.284 11.624 1598 CB THR 194 −6.59452.008 11.267 1599 OG1 THR 194 −6.679 51.178 12.419 1600 CG2 THR 194−5.879 51.224 10.172 1601 C THR 194 −6.628 54.102 12.643 1602 O THR 194−7.778 53.767 12.949 1603 N CYS 195 −6.038 55.184 13.127 1605 CA CYS 195−6.763 56.090 14.023 1606 CB CYS 195 −5.924 56.299 15.279 1607 SG CYS195 −5.598 54.819 16.262 1608 C CYS 195 −6.992 57.434 13.342 1609 O CYS195 −6.096 58.279 13.351 1610 N PRO 196 −8.127 57.602 12.686 1611 CA PRO196 −8.392 58.842 11.953 1612 CB PRO 196 −9.475 58.481 10.984 1613 CGPRO 196 −10.070 57.140 11.389 1614 CD PRO 196 −9.222 56.638 12.548 1615C PRO 196 −8.853 59.980 12.862 1616 O PRO 196 −9.020 59.801 14.074 1617N LYS 197 −8.872 61.162 12.264 1619 CA LYS 197 −9.517 62.397 12.769 1620CB LYS 197 −11.033 62.207 12.961 1621 CG LYS 197 −11.443 61.549 14.2781622 CD LYS 197 −12.955 61.388 14.391 1623 CE LYS 197 −13.667 62.73614.402 1624 NZ LYS 197 −15.125 62.567 14.529 1625 C LYS 197 −8.89063.026 14.018 1626 O LYS 197 −8.593 62.383 15.033 1627 N PRO 198 −8.63364.316 13.884 1628 CA PRO 198 −8.458 65.184 15.049 1629 CB PRO 198−8.097 66.522 14.486 1630 CG PRO 198 −8.299 66.495 12.979 1631 CD PRO198 −8.726 65.078 12.637 1632 C PRO 198 −9.747 65.276 15.861 1633 O PRO198 −10.852 65.161 15.319 1634 N ASP 199 −9.590 65.480 17.156 1636 CAASP 199 −10.746 65.583 18.054 1637 CB ASP 199 −11.299 64.182 18.311 1638CG ASP 199 −12.690 64.271 18.937 1639 OD1 ASP 199 −13.012 63.414 19.7451640 OD2 ASP 199 −13.391 65.220 18.610 1641 C ASP 199 −10.340 66.24319.372 1642 O ASP 199 −9.345 65.861 19.998 1643 N PHE 200 −11.089 67.25619.774 1645 CA PHE 200 −10.774 67.952 21.027 1646 CB PHE 200 −11.39769.344 20.991 1647 CG PHE 200 −11.066 70.229 22.194 1648 CD1 PHE 200−12.007 71.133 22.672 1649 CE1 PHE 200 −11.703 71.942 23.759 1650 CZ PHE200 −10.460 71.848 24.370 1651 CE2 PHE 200 −9.521 70.942 23.896 1652 CD2PHE 200 −9.825 70.134 22.809 1653 C PHE 200 −11.306 67.178 22.231 1654 OPHE 200 −12.516 67.115 22.468 1655 N ILE 201 −10.386 66.576 22.964 1657CA ILE 201 −10.736 65.876 24.200 1658 CB ILE 201 −10.063 64.506 24.1911659 CG2 ILE 201 −10.399 63.709 25.447 1660 CG1 ILE 201 −10.483 63.72322.950 1661 CD1 ILE 201 −9.845 62.339 22.914 1662 C ILE 201 −10.24666.740 25.359 1663 O ILE 201 −9.065 66.659 25.705 1664 N PRO 202 −11.19967.254 26.123 1665 CA PRO 202 −11.264 68.709 26.425 1666 CB PRO 202−12.547 68.886 27.179 1667 CG PRO 202 −13.369 67.615 27.069 1668 CD PRO202 −12.547 66.681 26.203 1669 C PRO 202 −10.116 69.368 27.205 1670 OPRO 202 −10.119 70.594 27.344 1671 N GLU 203 −9.144 68.616 27.689 1673CA GLU 203 −7.980 69.248 28.305 1674 CB GLU 203 −7.541 68.427 29.5101675 CG GLU 203 −8.638 68.361 30.568 1676 CD GLU 203 −8.914 69.74531.154 1677 OE1 GLU 203 −7.992 70.309 31.728 1678 OE2 GLU 203 −10.08070.111 31.188 1679 C GLU 203 −6.840 69.357 27.292 1680 O GLU 203 −5.87970.098 27.524 1681 N SER 204 −6.958 68.635 26.186 1683 CA SER 204 −5.95768.708 25.112 1684 CB SER 204 −4.875 67.658 25.356 1685 OG SER 204−4.087 68.080 26.463 1686 C SER 204 −6.560 68.499 23.722 1687 O SER 204−7.566 67.800 23.537 1688 N HIS 205 −5.935 69.132 22.745 1690 CA HIS 205−6.337 68.947 21.346 1691 CB HIS 205 −5.946 70.170 20.526 1692 CG HIS205 −6.757 71.416 20.809 1693 ND1 HIS 205 −7.917 71.758 20.219 1695 CE1HIS 205 −8.346 72.932 20.723 1696 NE2 HIS 205 −7.441 73.337 21.642 1697CD2 HIS 205 −6.454 72.414 21.706 1698 C HIS 205 −5.649 67.724 20.7601699 O HIS 205 −4.417 67.681 20.650 1700 N PHE 206 −6.445 66.735 20.3981702 CA PHE 206 −5.893 65.503 19.830 1703 CB PHE 206 −6.668 64.29120.349 1704 CG PHE 206 −6.362 63.842 21.780 1705 CD1 PHE 206 −5.79962.589 21.988 1706 CE1 PHE 206 −5.520 62.156 23.278 1707 CZ PHE 206−5.809 62.973 24.364 1708 CE2 PHE 206 −6.379 64.222 24.159 1709 CD2 PHE206 −6.659 64.653 22.869 1710 C PHE 206 −5.931 65.515 18.306 1711 O PHE206 −6.837 66.076 17.678 1712 N LEU 207 −4.886 64.954 17.730 1714 CA LEU207 −4.811 64.736 16.283 1715 CB LEU 207 −3.694 65.595 15.695 1716 CGLEU 207 −3.473 65.318 14.206 1717 CD1 LEU 207 −4.703 65.670 13.380 1718CD2 LEU 207 −2.256 66.066 13.678 1719 C LEU 207 −4.496 63.274 16.0091720 O LEU 207 −3.339 62.857 16.142 1721 N ARG 208 −5.513 62.488 15.7091723 CA ARG 208 −5.245 61.105 15.342 1724 CB ARG 208 −6.391 60.21815.792 1725 CG ARG 208 −6.334 59.973 17.292 1726 CD ARG 208 −7.43359.012 17.719 1727 NE ARG 208 −7.094 58.362 18.993 1728 CZ ARG 208−7.477 57.116 19.280 1729 NH1 ARG 208 −7.020 56.515 20.381 1730 NH2 ARG208 −8.225 56.435 18.408 1731 C ARG 208 −4.979 60.985 13.847 1732 O ARG208 −5.726 61.482 12.991 1733 N VAL 209 −3.809 60.437 13.581 1735 CA VAL209 −3.318 60.229 12.222 1736 CB VAL 209 −1.807 60.448 12.257 1737 CG1VAL 209 −1.179 60.279 10.881 1738 CG2 VAL 209 −1.470 61.820 12.826 1739C VAL 209 −3.628 58.806 11.779 1740 O VAL 209 −3.208 57.841 12.430 1741N PRO 210 −4.391 58.682 10.704 1742 CA PRO 210 −4.766 57.368 10.164 1743CB PRO 210 −5.846 57.657 9.167 1744 CG PRO 210 −5.951 59.161 8.962 1745CD PRO 210 −4.969 59.788 9.937 1746 C PRO 210 −3.598 56.647 9.489 1747 OPRO 210 −3.458 56.678 8.259 1748 N VAL 211 −2.783 55.981 10.289 1750 CAVAL 211 −1.663 55.217 9.746 1751 CB VAL 211 −0.511 55.251 10.742 1752CG1 VAL 211 0.701 54.528 10.172 1753 CG2 VAL 211 −0.144 56.683 11.1081754 C VAL 211 −2.077 53.773 9.505 1755 O VAL 211 −2.116 52.964 10.4371756 N ASN 212 −2.475 53.477 8.281 1758 CA ASN 212 −2.814 52.095 7.9451759 CB ASN 212 −3.391 52.062 6.531 1760 CG ASN 212 −3.885 50.667 6.1651761 OD1 ASN 212 −3.091 49.803 5.778 1762 ND2 ASN 212 −5.169 50.4386.371 1765 C ASN 212 −1.548 51.245 8.086 1766 O ASN 212 −0.436 51.7127.825 1767 N ASP 213 −1.721 50.020 8.552 1769 CA ASP 213 −0.585 49.1538.890 1770 CB ASP 213 −1.025 48.214 10.012 1771 CG ASP 213 −2.251 47.3979.602 1772 OD1 ASP 213 −3.355 47.845 9.884 1773 OD2 ASP 213 −2.05846.299 9.099 1774 C ASP 213 −0.023 48.339 7.717 1775 O ASP 213 0.93647.584 7.915 1776 N SER 214 −0.602 48.459 6.532 1778 CA SER 214 −0.05847.751 5.366 1779 CB SER 214 −1.032 47.848 4.198 1780 OG SER 214 −1.05149.200 3.758 1781 C SER 214 1.271 48.364 4.947 1782 O SER 214 1.47549.577 5.070 1783 N PHE 215 2.070 47.566 4.259 1785 CA PHE 215 3.41247.996 3.831 1786 CB PHE 215 4.265 46.755 3.594 1787 CG PHE 215 4.53345.953 4.865 1788 CD1 PHE 215 5.122 46.572 5.961 1789 CE1 PHE 215 5.36345.848 7.121 1790 CZ PHE 215 5.017 44.505 7.186 1791 CE2 PHE 215 4.43243.885 6.089 1792 CD2 PHE 215 4.190 44.609 4.928 1793 C PHE 215 3.43248.889 2.583 1794 O PHE 215 4.509 49.209 2.072 1795 N CYS 216 2.26449.299 2.113 1797 CA CYS 216 2.173 50.207 0.970 1798 CB CYS 216 1.00949.750 0.098 1799 SG CYS 216 1.098 48.041 −0.484 1800 C CYS 216 1.93951.654 1.414 1801 O CYS 216 1.796 52.544 0.568 1802 N GLU 217 1.88151.886 2.717 1804 CA GLU 217 1.550 53.228 3.222 1805 CB GLU 217 1.10653.114 4.672 1806 CG GLU 217 −0.102 52.197 4.791 1807 CD GLU 217 −1.24252.682 3.905 1808 OE1 GLU 217 −1.800 53.726 4.218 1809 OE2 GLU 217−1.568 51.975 2.962 1810 C GLU 217 2.691 54.239 3.122 1811 O GLU 2173.853 53.964 3.449 1812 N LYS 218 2.313 55.425 2.680 1814 CA LYS 2183.241 56.553 2.604 1815 CB LYS 218 3.047 57.226 1.251 1816 CG LYS 2184.097 58.293 0.979 1817 CD LYS 218 3.893 58.903 −0.401 1818 CE LYS 2184.921 59.989 −0.685 1819 NZ LYS 218 4.708 60.575 −2.016 1820 C LYS 2182.951 57.524 3.747 1821 O LYS 218 1.867 58.112 3.832 1822 N ILE 2193.941 57.698 4.604 1824 CA ILE 219 3.795 58.509 5.814 1825 CB ILE 2194.689 57.887 6.889 1826 CG2 ILE 219 4.646 58.672 8.196 1827 CG1 ILE 2194.278 56.442 7.150 1828 CD1 ILE 219 2.874 56.363 7.741 1829 C ILE 2194.179 59.962 5.549 1830 O ILE 219 3.709 60.860 6.256 1831 N LEU 2204.807 60.203 4.411 1833 CA LEU 220 5.187 61.570 4.006 1834 CB LEU 2205.902 61.485 2.664 1835 CG LEU 220 7.197 60.693 2.812 1836 CD1 LEU 2207.880 60.476 1.469 1837 CD2 LEU 220 8.152 61.375 3.786 1838 C LEU 2204.054 62.627 3.972 1839 O LEU 220 4.258 63.658 4.625 1840 N PRO 2212.870 62.399 3.400 1841 CA PRO 221 1.821 63.428 3.514 1842 CB PRO 2210.704 62.962 2.631 1843 CG PRO 221 1.033 61.587 2.075 1844 CD PRO 2212.409 61.245 2.614 1845 C PRO 221 1.313 63.659 4.948 1846 O PRO 2211.090 64.820 5.327 1847 N TRP 222 1.408 62.644 5.796 1849 CA TRP 2221.005 62.794 7.195 1850 CB TRP 222 0.689 61.429 7.794 1851 CG TRP 222−0.579 60.738 7.326 1852 CD1 TRP 222 −0.798 59.377 7.365 1853 NE1 TRP222 −2.052 59.126 6.897 1855 CE2 TRP 222 −2.671 60.262 6.545 1856 CZ2TRP 222 −3.934 60.525 6.025 1857 CH2 TRP 222 −4.315 61.827 5.753 1858CZ3 TRP 222 −3.442 62.886 5.998 1859 CE3 TRP 222 −2.173 62.631 6.5161860 CD2 TRP 222 −1.789 61.334 6.785 1861 C TRP 222 2.102 63.453 8.0221862 O TRP 222 1.792 64.136 9.003 1863 N LEU 223 3.320 63.447 7.503 1865CA LEU 223 4.414 64.182 8.136 1866 CB LEU 223 5.751 63.687 7.598 1867 CGLEU 223 6.030 62.250 8.017 1868 CD1 LEU 223 7.328 61.744 7.398 1869 CD2LEU 223 6.080 62.130 9.534 1870 C LEU 223 4.287 65.670 7.857 1871 O LEU223 4.537 66.471 8.762 1872 N ASP 224 3.635 66.014 6.760 1874 CA ASP 2243.373 67.424 6.462 1875 CB ASP 224 2.833 67.532 5.039 1876 CG ASP 2243.803 66.906 4.037 1877 OD1 ASP 224 4.995 67.150 4.166 1878 OD2 ASP 2243.324 66.265 3.110 1879 C ASP 224 2.341 67.975 7.445 1880 O ASP 2242.650 68.910 8.200 1881 N LYS 225 1.297 67.188 7.662 1883 CA LYS 2250.232 67.576 8.595 1884 CB LYS 225 −0.901 66.566 8.460 1885 CG LYS 225−2.020 66.846 9.457 1886 CD LYS 225 −3.065 65.735 9.450 1887 CE LYS 225−2.455 64.393 9.841 1888 NZ LYS 225 −3.481 63.338 9.885 1889 C LYS 2250.711 67.574 10.045 1890 O LYS 225 0.487 68.556 10.768 1891 N SER 2261.576 66.630 10.373 1893 CA SER 226 2.082 66.521 11.739 1894 CB SER 2262.653 65.123 11.947 1895 OG SER 226 3.777 64.952 11.096 1896 C SER 2263.139 67.575 12.064 1897 O SER 226 3.093 68.111 13.175 1898 N VAL 2273.843 68.085 11.064 1900 CA VAL 227 4.790 69.176 11.312 1901 CB VAL 2275.781 69.264 10.156 1902 CG1 VAL 227 6.560 70.574 10.189 1903 CG2 VAL227 6.732 68.074 10.160 1904 C VAL 227 4.050 70.497 11.469 1905 O VAL227 4.399 71.285 12.357 1906 N ASP 228 2.872 70.574 10.870 1908 CA ASP228 2.023 71.750 11.050 1909 CB ASP 228 0.871 71.685 10.049 1910 CG ASP228 1.379 71.634 8.610 1911 OD1 ASP 228 0.674 71.059 7.788 1912 OD2 ASP228 2.371 72.293 8.328 1913 C ASP 228 1.451 71.778 12.466 1914 O ASP 2281.635 72.777 13.174 1915 N PHE 229 1.066 70.611 12.959 1917 CA PHE 2290.464 70.509 14.295 1918 CB PHE 229 −0.196 69.139 14.388 1919 CG PHE 229−1.042 68.891 15.635 1920 CD1 PHE 229 −2.321 69.426 15.714 1921 CE1 PHE229 −3.105 69.190 16.836 1922 CZ PHE 229 −2.610 68.419 17.879 1923 CE2PHE 229 −1.331 67.886 17.802 1924 CD2 PHE 229 −0.546 68.122 16.680 1925C PHE 229 1.497 70.672 15.411 1926 O PHE 229 1.258 71.433 16.360 1927 NILE 230 2.706 70.191 15.170 1929 CA ILE 230 3.773 70.331 16.162 1930 CBILE 230 4.883 69.334 15.845 1931 CG2 ILE 230 6.064 69.525 16.786 1932CG1 ILE 230 4.393 67.897 15.934 1933 CD1 ILE 230 5.503 66.930 15.5401934 C ILE 230 4.354 71.741 16.165 1935 O ILE 230 4.669 72.265 17.2401936 N GLU 231 4.253 72.435 15.045 1938 CA GLU 231 4.761 73.803 14.9921939 CB GLU 231 5.053 74.155 13.540 1940 CG GLU 231 5.797 75.477 13.4191941 CD GLU 231 6.190 75.694 11.963 1942 OE1 GLU 231 7.262 75.238 11.5901943 OE2 GLU 231 5.381 76.247 11.231 1944 C GLU 231 3.760 74.781 15.5931945 O GLU 231 4.174 75.746 16.248 1946 N LYS 232 2.492 74.403 15.6051948 CA LYS 232 1.496 75.223 16.293 1949 CB LYS 232 0.103 74.842 15.8071950 CG LYS 232 −0.073 75.177 14.330 1951 CD LYS 232 −1.464 74.79413.839 1952 CE LYS 232 −1.738 73.313 14.067 1953 NZ LYS 232 −3.06572.927 13.561 1954 C LYS 232 1.594 75.037 17.802 1955 O LYS 232 1.61476.039 18.526 1956 N ALA 233 1.968 73.842 18.232 1958 CA ALA 233 2.16273.599 19.666 1959 CB ALA 233 2.174 72.096 19.894 1960 C ALA 233 3.47174.183 20.189 1961 O ALA 233 3.523 74.671 21.328 1962 N LYS 234 4.44974.311 19.308 1964 CA LYS 234 5.730 74.895 19.695 1965 CB LYS 234 6.79874.373 18.739 1966 CG LYS 234 8.196 74.824 19.143 1967 CD LYS 234 9.26274.205 18.247 1968 CE LYS 234 10.664 74.616 18.684 1969 NZ LYS 23411.685 74.023 17.805 1970 C LYS 234 5.698 76.421 19.680 1971 O LYS 2346.348 77.047 20.526 1972 N ALA 235 4.821 76.998 18.875 1974 CA ALA 2354.715 78.460 18.830 1975 CB ALA 235 4.370 78.881 17.406 1976 C ALA 2353.665 79.004 19.794 1977 O ALA 235 3.742 80.169 20.204 1978 N SER 2362.745 78.152 20.212 1980 CA SER 236 1.736 78.568 21.189 1981 CB SER 2360.418 77.878 20.862 1982 OG SER 236 0.053 78.240 19.537 1983 C SER 2362.143 78.213 22.613 1984 O SER 236 1.558 78.748 23.563 1985 N ASN 2373.216 77.445 22.738 1987 CA ASN 237 3.701 76.962 24.037 1988 CB ASN 2374.146 78.125 24.922 1989 CG ASN 237 5.345 78.825 24.288 1990 OD1 ASN 2376.427 78.236 24.164 1991 ND2 ASN 237 5.136 80.064 23.878 1994 C ASN 2372.629 76.119 24.710 1995 O ASN 237 1.961 76.542 25.662 1996 N GLY 2382.451 74.938 24.145 1998 CA GLY 238 1.461 73.974 24.628 1999 C GLY 2381.837 72.589 24.119 2000 O GLY 238 0.995 71.895 23.529 2001 N CYS 2392.982 72.139 24.612 2003 CA CYS 239 3.725 70.941 24.168 2004 CB CYS 2394.387 70.323 25.393 2005 SG CYS 239 5.410 71.424 26.398 2006 C CYS 2392.907 69.848 23.494 2007 O CYS 239 1.839 69.447 23.977 2008 N VAL 2403.415 69.384 22.368 2010 CA VAL 240 2.763 68.277 21.673 2011 CB VAL 2402.771 68.557 20.173 2012 CG1 VAL 240 4.169 68.889 19.679 2013 CG2 VAL240 2.162 67.419 19.362 2014 C VAL 240 3.443 66.949 21.995 2015 O VAL240 4.670 66.816 21.910 2016 N LEU 241 2.652 66.024 22.507 2018 CA LEU241 3.128 64.656 22.723 2019 CB LEU 241 2.402 64.062 23.927 2020 CG LEU241 2.804 62.611 24.186 2021 CD1 LEU 241 4.297 62.483 24.460 2022 CD2LEU 241 2.008 62.020 25.342 2023 C LEU 241 2.830 63.820 21.484 2024 OLEU 241 1.660 63.604 21.150 2025 N VAL 242 3.871 63.411 20.782 2027 CAVAL 242 3.692 62.559 19.601 2028 CB VAL 242 4.743 62.918 18.560 2029 CG1VAL 242 4.575 62.071 17.303 2030 CG2 VAL 242 4.664 64.399 18.214 2031 CVAL 242 3.825 61.097 20.009 2032 O VAL 242 4.932 60.549 20.059 2033 NHIS 243 2.688 60.475 20.264 2035 CA HIS 243 2.668 59.131 20.844 2036 CBHIS 243 1.799 59.180 22.099 2037 CG HIS 243 1.992 58.018 23.055 2038 ND1HIS 243 1.427 56.797 22.981 2040 CE1 HIS 243 1.853 56.044 24.014 2041NE2 HIS 243 2.686 56.804 24.757 2042 CD2 HIS 243 2.782 58.023 24.1802043 C HIS 243 2.125 58.080 19.878 2044 O HIS 243 1.027 58.217 19.3132045 N CYS 244 2.876 57.004 19.731 2047 CA CYS 244 2.405 55.873 18.9272048 CB CYS 244 3.325 55.672 17.734 2049 SG CYS 244 2.828 54.350 16.6082050 C CYS 244 2.344 54.595 19.769 2051 O CYS 244 3.374 54.023 20.1332052 N LEU 245 1.138 54.053 19.864 2054 CA LEU 245 0.892 52.873 20.7112055 CB LEU 245 −0.610 52.805 20.966 2056 CG LEU 245 −1.097 53.96021.831 2057 CD1 LEU 245 −2.605 54.149 21.705 2058 CD2 LEU 245 −0.69053.751 23.285 2059 C LEU 245 1.322 51.573 20.043 2060 O LEU 245 1.55450.565 20.724 2061 N ALA 246 1.562 51.635 18.742 2063 CA ALA 246 1.99150.460 17.991 2064 CB ALA 246 1.425 50.559 16.582 2065 C ALA 246 3.51150.340 17.913 2066 O ALA 246 4.021 49.300 17.484 2067 N GLY 247 4.21851.351 18.387 2069 CA GLY 247 5.676 51.309 18.318 2070 C GLY 247 6.28952.696 18.244 2071 O GLY 247 5.753 53.597 17.584 2072 N ILE 248 7.54152.754 18.667 2074 CA ILE 248 8.309 54.008 18.678 2075 CB ILE 248 9.54453.780 19.556 2076 CG2 ILE 248 10.278 52.506 19.154 2077 CG1 ILE 24810.506 54.965 19.565 2078 CD1 ILE 248 9.877 56.213 20.175 2079 C ILE 2488.726 54.464 17.273 2080 O ILE 248 8.894 55.670 17.066 2081 N SER 2498.548 53.599 16.286 2083 CA SER 249 8.948 53.904 14.914 2084 CB SER 2498.702 52.668 14.063 2085 OG SER 249 9.535 51.626 14.547 2086 C SER 2498.172 55.074 14.323 2087 O SER 249 8.802 56.077 13.979 2088 N ARG 2506.862 55.093 14.508 2090 CA ARG 250 6.064 56.145 13.870 2091 CB ARG 2504.629 55.657 13.752 2092 CG ARG 250 4.576 54.344 12.985 2093 CD ARG 2503.153 53.813 12.894 2094 NE ARG 250 3.111 52.544 12.152 2095 CZ ARG 2502.123 51.662 12.300 2096 NH1 ARG 250 1.149 51.902 13.177 2097 NH2 ARG250 2.119 50.533 11.588 2098 C ARG 250 6.103 57.457 14.646 2099 O ARG250 6.185 58.525 14.025 2100 N SER 251 6.352 57.371 15.943 2102 CA SER251 6.427 58.595 16.746 2103 CB SER 251 6.123 58.278 18.210 2104 OG SER251 7.062 57.334 18.708 2105 C SER 251 7.800 59.249 16.599 2106 O SER251 7.883 60.478 16.465 2107 N ALA 252 8.798 58.429 16.309 2109 CA ALA252 10.142 58.935 16.056 2110 CB ALA 252 11.137 57.805 16.273 2111 C ALA252 10.268 59.440 14.629 2112 O ALA 252 10.911 60.471 14.414 2113 N THR253 9.453 58.905 13.736 2115 CA THR 253 9.440 59.380 12.352 2116 CB THR253 8.625 58.406 11.508 2117 OG1 THR 253 9.334 57.178 11.471 2118 CG2THR 253 8.463 58.891 10.072 2119 C THR 253 8.825 60.768 12.261 2120 OTHR 253 9.421 61.652 11.633 2121 N ILE 254 7.834 61.030 13.097 2123 CAILE 254 7.224 62.358 13.106 2124 CB ILE 254 5.853 62.247 13.757 2125 CG2ILE 254 5.221 63.621 13.928 2126 CG1 ILE 254 4.952 61.341 12.926 2127CD1 ILE 254 3.547 61.268 13.504 2128 C ILE 254 8.083 63.382 13.844 2129O ILE 254 8.256 64.497 13.334 2130 N ALA 255 8.826 62.936 14.845 2132 CAALA 255 9.707 63.860 15.567 2133 CB ALA 255 10.123 63.216 16.884 2134 CALA 255 10.947 64.212 14.747 2135 O ALA 255 11.255 65.401 14.590 2136 NILE 256 11.461 63.238 14.014 2138 CA ILE 256 12.631 63.468 13.167 2139CB ILE 256 13.209 62.118 12.759 2140 CG2 ILE 256 14.277 62.283 11.6892141 CG1 ILE 256 13.786 61.378 13.958 2142 CD1 ILE 256 14.249 59.98313.557 2143 C ILE 256 12.267 64.267 11.923 2144 O ILE 256 12.977 65.23211.616 2145 N ALA 257 11.057 64.080 11.417 2147 CA ALA 257 10.613 64.85010.253 2148 CB ALA 257 9.383 64.182 9.656 2149 C ALA 257 10.271 66.28710.625 2150 O ALA 257 10.563 67.203 9.842 2151 N TYR 258 9.902 66.50411.877 2153 CA TYR 258 9.688 67.871 12.334 2154 CB TYR 258 8.931 67.89013.654 2155 CG TYR 258 8.683 69.315 14.140 2156 CD1 TYR 258 7.806 70.13013.437 2157 CE1 TYR 258 7.576 71.432 13.860 2158 CZ TYR 258 8.229 71.91714.984 2159 OH TYR 258 7.935 73.179 15.447 2160 CE2 TYR 258 9.120 71.11115.680 2161 CD2 TYR 258 9.351 69.809 15.254 2162 C TYR 258 11.014 68.59012.517 2163 O TYR 258 11.150 69.697 11.992 2164 N ILE 259 12.039 67.88912.974 2166 CA ILE 259 13.350 68.527 13.150 2167 CB ILE 259 14.22667.598 13.982 2168 CG2 ILE 259 15.612 68.193 14.181 2169 CG1 ILE 25913.593 67.313 15.337 2170 CD1 ILE 259 14.455 66.354 16.149 2171 C ILE259 14.024 68.802 11.804 2172 O ILE 259 14.536 69.912 11.586 2173 N MET260 13.763 67.933 10.841 2175 CA MET 260 14.296 68.114 9.490 2176 CB MET260 14.010 66.853 8.683 2177 CG MET 260 14.786 65.655 9.213 2178 SD MET260 14.376 64.077 8.439 2179 CE MET 260 14.817 64.490 6.740 2180 C MET260 13.669 69.305 8.776 2181 O MET 260 14.412 70.145 8.258 2182 N LYS261 12.377 69.514 8.963 2184 CA LYS 261 11.704 70.625 8.281 2185 CB LYS261 10.258 70.188 8.068 2186 CG LYS 261 9.447 71.160 7.220 2187 CD LYS261 8.056 70.592 6.960 2188 CE LYS 261 7.168 71.552 6.179 2189 NZ LYS261 5.823 70.979 5.996 2190 C LYS 261 11.759 71.942 9.068 2191 O LYS 26111.736 73.020 8.463 2192 N ARG 262 12.016 71.854 10.362 2194 CA ARG 26212.046 73.043 11.222 2195 CB ARG 262 11.654 72.575 12.623 2196 CG ARG262 11.618 73.676 13.676 2197 CD ARG 262 10.536 74.711 13.393 2198 NEARG 262 10.381 75.612 14.546 2199 CZ ARG 262 10.979 76.801 14.652 2200NH1 ARG 262 11.729 77.267 13.651 2201 NH2 ARG 262 10.793 77.542 15.7472202 C ARG 262 13.424 73.698 11.278 2203 O ARG 262 13.521 74.929 11.3492204 N MET 263 14.473 72.899 11.192 2206 CA MET 263 15.819 73.469 11.2922207 CB MET 263 16.469 72.887 12.542 2208 CG MET 263 17.636 73.73713.033 2209 SD MET 263 17.219 75.450 13.433 2210 CE MET 263 15.94175.154 14.678 2211 C MET 263 16.657 73.189 10.043 2212 O MET 263 17.82973.583 9.971 2213 N ASP 264 16.027 72.567 9.057 2215 CA ASP 264 16.68372.159 7.803 2216 CB ASP 264 17.184 73.378 7.033 2217 CG ASP 264 17.60872.969 5.626 2218 OD1 ASP 264 16.730 72.867 4.781 2219 OD2 ASP 26418.798 72.781 5.413 2220 C ASP 264 17.817 71.187 8.110 2221 O ASP 26419.007 71.511 8.005 2222 N MET 265 17.422 70.008 8.554 2224 CA MET 26518.393 68.987 8.964 2225 CB MET 265 18.257 68.747 10.465 2226 CG MET 26518.659 69.991 11.252 2227 SD MET 265 18.493 69.900 13.049 2228 CE MET265 19.643 68.553 13.395 2229 C MET 265 18.210 67.675 8.211 2230 O MET265 17.121 67.342 7.729 2231 N SER 266 19.305 66.946 8.097 2233 CA SER266 19.275 65.618 7.480 2234 CB SER 266 20.700 65.160 7.195 2235 OG SER266 21.287 64.788 8.437 2236 C SER 266 18.652 64.614 8.435 2237 O SER266 18.636 64.840 9.652 2238 N LEU 267 18.352 63.437 7.910 2240 CA LEU267 17.824 62.339 8.726 2241 CB LEU 267 17.490 61.188 7.778 2242 CG LEU267 17.424 59.902 8.513 2243 CD1 LEU 267 15.935 60.100 9.443 2244 CD2LEU 267 16.846 58.772 7.531 2245 C LEU 267 18.847 61.879 9.763 2246 OLEU 267 18.504 61.759 10.946 2247 N ASP 268 20.114 61.926 9.382 2249 CAASP 268 21.202 61.569 10.295 2250 CB ASP 268 22.526 61.630 9.537 2251 CGASP 268 22.457 60.802 8.256 2252 OD1 ASP 268 22.183 61.394 7.220 2253OD2 ASP 268 22.622 59.595 8.342 2254 C ASP 268 21.267 62.538 11.466 2255O ASP 268 21.062 62.115 12.612 2256 N GLU 269 21.285 63.832 11.175 2258CA GLU 269 21.395 64.837 12.234 2259 CB GLU 269 21.624 66.181 11.5602260 CG GLU 269 22.967 66.232 10.846 2261 CD GLU 269 22.978 67.413 9.8872262 OE1 GLU 269 24.047 67.959 9.656 2263 OE2 GLU 269 21.942 67.6199.266 2264 C GLU 269 20.146 64.939 13.109 2265 O GLU 269 20.283 65.06414.331 2266 N ALA 270 18.980 64.678 12.544 2268 CA ALA 270 17.754 64.77013.334 2269 CB ALA 270 16.575 64.932 12.387 2270 C ALA 270 17.557 63.54814.223 2271 O ALA 270 17.222 63.718 15.402 2272 N TYR 271 18.038 62.39513.783 2274 CA TYR 271 17.934 61.198 14.621 2275 CB TYR 271 18.04459.951 13.750 2276 CG TYR 271 17.886 58.641 14.520 2277 CD1 TYR 27116.999 58.562 15.588 2278 CE1 TYR 271 16.866 57.376 16.297 2279 CZ TYR271 17.615 56.266 15.930 2280 OH TYR 271 17.553 55.121 16.695 2281 CE2TYR 271 18.486 56.333 14.851 2282 CD2 TYR 271 18.619 57.522 14.143 2283C TYR 271 19.026 61.187 15.688 2284 O TYR 271 18.753 60.800 16.832 2285N ARG 272 20.141 61.840 15.408 2287 CA ARG 272 21.185 61.963 16.426 2288CB ARG 272 22.523 62.222 15.748 2289 CG ARG 272 22.978 60.971 15.0062290 CD ARG 272 24.355 61.137 14.378 2291 NE ARG 272 24.332 62.08213.251 2292 CZ ARG 272 25.283 62.995 13.050 2293 NH1 ARG 272 26.25163.159 13.954 2294 NH2 ARG 272 25.226 63.792 11.982 2295 C ARG 27220.870 63.054 17.446 2296 O ARG 272 21.251 62.913 18.614 2297 N PHE 27319.981 63.968 17.093 2299 CA PHE 273 19.504 64.947 18.068 2300 CB PHE273 18.958 66.157 17.319 2301 CG PHE 273 18.551 67.310 18.228 2302 CD1PHE 273 19.525 68.151 18.749 2303 CE1 PHE 273 19.163 69.201 19.583 2304CZ PHE 273 17.826 69.409 19.896 2305 CE2 PHE 273 16.852 68.568 19.3752306 CD2 PHE 273 17.214 67.519 18.541 2307 C PHE 273 18.411 64.33318.942 2308 O PHE 273 18.388 64.572 20.158 2309 N VAL 274 17.705 63.35718.394 2311 CA VAL 274 16.709 62.636 19.187 2312 CB VAL 274 15.75761.882 18.267 2313 CG1 VAL 274 14.789 61.044 19.085 2314 CG2 VAL 27414.982 62.838 17.371 2315 C VAL 274 17.376 61.662 20.155 2316 O VAL 27417.024 61.682 21.340 2317 N LYS 275 18.504 61.086 19.759 2319 CA LYS 27519.266 60.236 20.687 2320 CB LYS 275 20.178 59.287 19.921 2321 CG LYS275 19.391 58.332 19.034 2322 CD LYS 275 20.254 57.154 18.596 2323 CELYS 275 21.503 57.599 17.842 2324 NZ LYS 275 21.154 58.252 16.572 2325 CLYS 275 20.114 61.051 21.663 2326 O LYS 275 20.486 60.545 22.727 2327 NGLU 276 20.246 62.339 21.394 2329 CA GLU 276 20.914 63.252 22.320 2330CB GLU 276 21.337 64.477 21.515 2331 CG GLU 276 22.061 65.526 22.3482332 CD GLU 276 22.358 66.739 21.474 2333 OE1 GLU 276 23.388 66.72620.813 2334 OE2 GLU 276 21.508 67.616 21.410 2335 C GLU 276 19.97163.665 23.452 2336 O GLU 276 20.433 64.057 24.530 2337 N LYS 277 18.67363.516 23.237 2339 CA LYS 277 17.717 63.755 24.320 2340 CB LYS 27716.526 64.542 23.781 2341 CG LYS 277 16.945 65.900 23.219 2342 CD LYS277 17.605 66.783 24.277 2343 CE LYS 277 16.634 67.198 25.380 2344 NZLYS 277 15.588 68.093 24.861 2345 C LYS 277 17.254 62.431 24.929 2346 OLYS 277 16.857 62.382 26.099 2347 N ARG 278 17.358 61.365 24.151 2349 CAARG 278 17.092 60.016 24.661 2350 CB ARG 278 15.598 59.737 24.629 2351CG ARG 278 15.286 58.490 25.446 2352 CD ARG 278 15.671 58.702 26.9052353 NE ARG 278 15.521 57.462 27.679 2354 CZ ARG 278 14.551 57.26128.575 2355 NH1 ARG 278 13.636 58.207 28.797 2356 NH2 ARG 278 14.49756.109 29.247 2357 C ARG 278 17.788 58.953 23.817 2358 O ARG 278 17.25358.513 22.791 2359 N PRO 279 18.865 58.407 24.357 2360 CA PRO 279 19.69557.439 23.624 2361 CB PRO 279 20.985 57.409 24.383 2362 CG PRO 27920.810 58.144 25.704 2363 CD PRO 279 19.413 58.737 25.675 2364 C PRO 27919.119 56.017 23.523 2365 O PRO 279 19.740 55.157 22.892 2366 N THR 28017.952 55.772 24.103 2368 CA THR 280 17.367 54.423 24.084 2369 CB THR280 16.712 54.130 25.429 2370 OG1 THR 280 15.545 54.930 25.544 2371 CG2THR 280 17.641 54.431 26.600 2372 C THR 280 16.317 54.248 22.985 2373 OTHR 280 15.642 53.212 22.948 2374 N ILE 281 16.116 55.264 22.160 2376 CAILE 281 15.115 55.153 21.093 2377 CB ILE 281 14.813 56.551 20.551 2378CG2 ILE 281 16.083 57.286 20.142 2379 CG1 ILE 281 13.837 56.482 19.3862380 CD1 ILE 281 13.684 57.828 18.700 2381 C ILE 281 15.584 54.23019.963 2382 O ILE 281 16.686 54.379 19.424 2383 N SER 282 14.748 53.25519.639 2385 CA SER 282 15.037 52.375 18.497 2386 CB SER 282 15.53751.035 19.031 2387 OG SER 282 14.601 50.550 19.986 2388 C SER 282 13.82652.177 17.575 2389 O SER 282 13.052 51.226 17.737 2390 N PRO 283 13.66253.083 16.622 2391 CA PRO 283 12.697 52.888 15.538 2392 CB PRO 28312.593 54.226 14.873 2393 CG PRO 283 13.738 55.102 15.354 2394 CD PRO283 14.466 54.288 16.410 2395 C PRO 283 13.187 51.838 14.543 2396 O PRO283 14.376 51.503 14.508 2397 N ASN 284 12.251 51.247 13.821 2399 CA ASN284 12.632 50.318 12.752 2400 CB ASN 284 11.479 49.389 12.358 2401 CGASN 284 10.260 50.105 11.771 2402 OD1 ASN 284 10.370 51.002 10.925 2403ND2 ASN 284 9.097 49.598 12.139 2406 C ASN 284 13.162 51.088 11.546 2407O ASN 284 12.869 52.277 11.360 2408 N PHE 285 13.795 50.352 10.650 2410CA PHE 285 14.449 50.943 9.478 2411 CB PHE 285 15.557 49.993 9.015 2412CG PHE 285 15.106 48.601 8.557 2413 CD1 PHE 285 14.778 48.385 7.223 2414CE1 PHE 285 14.370 47.127 6.801 2415 CZ PHE 285 14.299 46.079 7.709 2416CE2 PHE 285 14.642 46.288 9.039 2417 CD2 PHE 285 15.051 47.546 9.4612418 C PHE 285 13.501 51.228 8.310 2419 O PHE 285 13.902 51.921 7.3652420 N ASN 286 12.221 50.940 8.483 2422 CA ASN 286 11.293 50.967 7.3492423 CB ASN 286 10.018 50.217 7.729 2424 CG ASN 286 10.255 48.757 8.1322425 OD1 ASN 286 11.362 48.209 8.051 2426 ND2 ASN 286 9.172 48.131 8.5582429 C ASN 286 10.913 52.399 7.003 2430 O ASN 286 10.858 52.770 5.8222431 N PHE 287 10.922 53.243 8.020 2433 CA PHE 287 10.572 54.648 7.8252434 CB PHE 287 9.917 55.164 9.096 2435 CG PHE 287 8.607 54.455 9.4232436 CD1 PHE 287 7.497 54.649 8.611 2437 CE1 PHE 287 6.305 53.997 8.8972438 CZ PHE 287 6.223 53.152 9.996 2439 CE2 PHE 287 7.331 52.962 10.8102440 CD2 PHE 287 8.523 53.614 10.524 2441 C PHE 287 11.771 55.518 7.4572442 O PHE 287 11.567 56.621 6.935 2443 N LEU 288 12.963 54.939 7.4502445 CA LEU 288 14.158 55.729 7.142 2446 CB LEU 288 15.388 54.990 7.6572447 CG LEU 288 15.341 54.812 9.171 4448 CD1 LEU 288 16.527 53.988 9.6582449 CD2 LEU 288 15.301 56.158 9.888 2450 C LEU 288 14.283 55.970 5.6402451 O LEU 288 14.716 57.055 5.235 2452 N GLY 289 13.636 55.116 4.8622454 CA GLY 289 13.570 55.303 3.409 2455 C GLY 289 12.730 56.528 3.0512456 O GLY 289 13.199 57.412 2.320 2457 N GLN 290 11.610 56.689 3.7422459 CA GLN 290 10.724 57.815 3.450 2460 CB GLN 290 9.321 57.510 3.9692461 CG GLN 290 8.737 56.292 3.258 2462 CD GLN 290 7.268 56.087 3.6252463 OE1 GLN 290 6.621 56.979 4.188 2464 NE2 GLN 290 6.741 54.941 3.2262467 C GLN 290 11.251 59.111 4.059 2468 O GLN 290 11.137 60.156 3.4102469 N LEU 291 12.083 59.002 5.082 2471 CA LEU 291 12.705 60.197 5.6592472 CB LEU 291 13.154 59.874 7.076 2473 CG LEU 291 11.952 59.682 7.9952474 CD1 LEU 291 12.369 59.130 9.352 2475 CD2 LEU 291 11.174 60.9848.153 2476 C LEU 291 13.883 60.693 4.819 2477 O LEU 291 14.070 61.9114.708 2478 N LEU 292 14.461 59.810 4.019 2480 CA LEU 292 15.504 60.2263.077 2481 CB LEU 292 16.303 59.003 2.647 2482 CG LEU 292 17.192 58.4973.774 2483 CD1 LEU 292 17.823 57.157 3.415 2484 CD2 LEU 292 18.26359.527 4.125 2485 C LEU 292 14.901 60.893 1.846 2486 O LEU 292 15.44461.896 1.366 2487 N ALA 293 13.677 60.511 1.516 2489 CA ALA 293 12.96761.156 0.406 2490 CB ALA 293 11.885 60.209 −0.097 2491 C ALA 293 12.33362.474 0.849 2492 O ALA 293 12.334 63.450 0.088 2493 N TYR 294 12.07262.569 2.144 2495 CA TYR 294 11.563 63.800 2.760 2496 CB TYR 294 11.08163.422 4.159 2497 CG TYR 294 10.455 64.529 5.006 2498 CD1 TYR 294 11.23765.246 5.903 2499 CE1 TYR 294 10.662 66.235 6.689 2500 CZ TYR 294 9.30466.499 6.583 2501 OH TYR 294 8.709 67.371 7.468 2502 CE2 TYR 294 8.51965.788 5.685 2503 CD2 TYR 294 9.097 64.800 4.897 2504 C TYR 294 12.66464.851 2.866 2505 O TYR 294 12.406 66.038 2.635 2506 N GLU 295 13.90064.378 2.912 2508 CA GLU 295 15.067 65.260 2.961 2509 CB GLU 295 16.23964.422 3.468 2510 CG GLU 295 17.511 65.241 3.650 2511 CD GLU 295 18.64164.341 4.132 2512 OE1 GLU 295 18.369 63.478 4.959 2513 OE2 GLU 29519.781 64.615 3.783 2514 C GLU 295 15.419 65.847 1.591 2515 O GLU 29516.150 66.843 1.517 2516 N LYS 296 14.816 65.332 0.532 2518 CA LYS 29615.134 65.839 −0.801 2519 CB LYS 296 15.062 64.667 −1.774 2520 CG LYS296 15.672 65.011 −3.128 2521 CD LYS 296 15.732 63.787 −4.036 2522 CELYS 296 16.437 64.105 −5.350 2523 NZ LYS 296 16.548 62.904 −6.193 2524 CLYS 296 14.188 66.967 −1.226 2525 O LYS 296 14.530 67.756 −2.117 2526 NLYS 297 13.087 67.141 −0.512 2528 CA LYS 297 12.156 68.219 −0.869 2529CB LYS 297 10.726 67.688 −0.879 2530 CG LYS 297 9.742 68.783 −1.284 2531CD LYS 297 10.067 69.352 −2.661 2532 CE LYS 297 9.246 70.605 −2.941 2533NZ LYS 297 9.576 71.679 −1.989 2534 C LYS 297 12.277 69.410 0.082 2535 OLYS 297 11.553 69.526 1.078 2536 N ILE 298 13.195 70.299 −0.254 2538 CAILE 298 13.370 71.527 0.525 2539 CB ILE 298 14.864 71.718 0.786 2540 CG2ILE 298 15.137 72.947 1.649 2541 CG1 ILE 298 15.440 70.479 1.463 2542CD1 ILE 298 16.919 70.658 1.788 2543 C ILE 298 12.767 72.723 −0.217 2544O ILE 298 12.943 72.876 −1.432 2545 N LYS 299 11.991 73.501 0.521 2547CA LYS 299 11.353 74.724 0.012 2548 CB LYS 299 10.719 75.409 1.225 2549CG LYS 299 10.135 76.796 0.945 2550 CD LYS 299 8.667 76.777 0.516 2551CE LYS 299 8.426 76.135 −0.845 2552 NZ LYS 299 6.997 76.161 −1.188 2553C LYS 299 12.334 75.698 −0.646 2554 O LYS 299 13.275 76.187 −0.010

1. An isolated protein comprising a polypeptide selected from the groupconsisting of: (a) the polypeptide of amino acids 1 to 665 of SEQ ID NO:109; and (b) the polypeptide of amino acids 2 to 665 of SEQ ID NO: 109.2. The isolated protein of claim 1, wherein said polypeptide is (a). 3.The isolated protein of claim 1, wherein said polypeptide is (b).
 4. Theisolated protein of claim 1 wherein said polypeptide further comprises aheterologous polypeptide.
 5. The isolated protein of claim 4 whereinsaid heterologous polypeptide is the Fc domain of immunoglobulin.
 6. Anisolated protein comprising a polypeptide encoded by the cDNA cloneselected from the group consisting of: (a) the polypeptide encoded bythe cDNA clone contained in plasmid RET31 in ATCC Deposit No. PTA-3434;and (b) the polypeptide encoded by the cDNA clone contained in plasmidBMY_HPP5 in ATCC Deposit No. PTA-2966.
 7. An isolated protein comprisingthe polypeptide of amino acids 2 to 665 of SEQ ID NO:109 with amino acidsubstitutions at amino acid residue 180, at amino acid residue 193, atamino acid residue 293, and at amino acid residue 315, wherein thesubstitute amino acid at amino acid residue 180 is methionine, thesubstitute amino acid at amino acid residue 193 is asparagine, thesubstitute amino acid at amino acid residue 293 is alanine, and thesubstitute amino acid at amino acid residue 315 is proline, and whereinsaid polypeptide has phosphatase activity, or is catalytically inactiveyet retains ability to bind to a phosphoprotein substrate.
 8. Anisolated protein comprising the polypeptide of amino acids 2 to 665 ofSEQ ID NO:109 with amino acid substitutions at amino acid residue 5, atamino acid residue 180, at amino acid residue 193, at amino acid residue284, at amino acid residue 302, and at amino acid residue 584, whereinthe substitute amino acid at amino acid residue 5 represents an aminoacid deletion at this position, the substitute amino acid at amino acidresidue 180 is methionine, the substitute amino acid at amino acidresidue 193 is asparagine, the substitute amino acid at amino acidresidue 284 is serine, the substitute amino acid at amino acid residue302 is alanine, and the substitute amino acid at amino acid residue 584is arginine, and wherein said polypeptide has phosphatase activity. 9.An isolated protein comprising the polypeptide of amino acids 2 to 665of SEQ ID NO:109 with amino acid substitutions at amino acid residue 5,at amino acid residue 6, at amino acid residue 180, at amino acidresidue 193, and at amino acid residue 284, wherein the substitute aminoacid at amino acid residue 5 is isoleucine, the substitute amino acid atamino acid residue 6 is valine, the substitute amino acid at amino acidresidue 180 is methionine, the substitute amino acid at amino acidresidue 193 is asparagine, and the substitute amino acid at aminoresidue 284 is serine, and wherein said polypeptide has phosphataseactivity.
 10. An isolated protein comprising amino acids 1 to 302 of SEQID NO:109.
 11. An isolated protein comprising amino acids 2 to 302 ofSEQ ID NO:109.
 12. An isolated protein comprising a polypeptideconsisting of at least 473 contiguous amino acids of SEQ ID NO:109,wherein said polypeptide has phosphatase activity.
 13. An isolatedprotein comprising the polypeptide of amino acids 1 to 302 of SEQ IDNO:109 with amino acid substitutions at amino acid residue 5, at aminoacid residue 6, at amino acid residue 180, at amino acid residue 193,and at amino acid residue 284, wherein the substitute amino acid atamino acid residue 5 is isoleucine, the substitute amino acid at aminoacid residue 6 is valine, the substitute amino acid at amino acidresidue 180 is methionine, the substitute amino acid at amino acidresidue 193 is asparagine, and the substitute amino acid at aminoresidue 284 is serine, wherein said polypeptide has phosphataseactivity.
 14. An isolated protein comprising the polypeptide of aminoacids 2 to 302 of SEQ ID NO:109 with amino acid substitutions at aminoacid residue 5, at amino acid residue 180, at amino acid residue 193, atamino acid residue 284, and at amino acid residue 302, wherein thesubstitute amino acid at amino acid residue 5 represents an amino aciddeletion at this position, the substitute amino acid at amino acidresidue 180 is methionine, the substitute amino acid at amino acidresidue 193 is asparagine, the substitute amino acid at amino residue284 is serine, and the substitute amino acid at amino acid residue 302is alanine, wherein said polypeptide has phosphatase activity.
 15. Anisolated protein comprising the polypeptide of amino acids 2 to 302 ofSEQ ID NO:109 with amino acid substitutions at amino acid residue 180,at amino acid residue 193, and at amino acid residue 293, wherein thesubstitute amino acid at amino acid residue 180 is methionine, thesubstitute amino acid at amino acid residue 193 is asparagine, and thesubstitute amino acid at amino residue 293 is alanine, wherein saidpolypeptide has phosphatase activity.
 16. An isolated protein producedby a method comprising: (a) culturing an isolated recombinant host cellcomprising a vector comprising the coding region encoding the protein ofclaim 1 under conditions such that the protein of claim 1 is expressed;and (b) recovering said polypeptide.